The Interactive Fly

Zygotically transcribed genes

Apoptosis and Autophagy

  • Cell death regulation in Drosophila: Conservation of mechanism and unique insights
  • Dying cells protect survivors from radiation-induced cell death in Drosophila
  • Effects of cell death-induced proliferation on a cell competition system
  • Transiently "Undead" Enterocytes Mediate Homeostatic Tissue Turnover in the Adult Drosophila Midgut
  • Tie-mediated signal from apoptotic cells protects stem cells in Drosophila melanogaster
  • A dual role of lola in Drosophila ovary development: regulating stem cell niche establishment and repressing apoptosis
  • Non-apoptotic caspase activation ensures the homeostasis of ovarian somatic stem cells
  • Analysis of the function of apoptosis during imaginal wing disc regeneration in Drosophila melanogaster
  • 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
  • In vivo biosensor tracks non-apoptotic caspase activity in Drosophila
  • Caspases maintain tissue integrity by an apoptosis-independent inhibition of cell migration and invasion
  • 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
  • Trans-generational transmission of altered phenotype resulting from flubendiamide-induced changes in apoptosis in larval imaginal discs of Drosophila melanogaster
  • Plasma membrane localization of apoptotic caspases for non-apoptotic functions
  • Live cell tracking of macrophage efferocytosis during Drosophila embryo development in vivo
  • Proto-pyroptosis: An ancestral origin for mammalian inflammatory cell death mechanism in Drosophila melanogaster
  • Evidence for a novel function of Awd in maintenance of genomic stability
  • NCBP2 modulates neurodevelopmental defects of the 3q29 deletion in Drosophila and Xenopus laevis models
  • A comprehensive in vivo screen for anti-apoptotic miRNAs indicates broad capacities for oncogenic synergy
  • Effects of cadmium on oxidative stress and cell apoptosis in Drosophila melanogaster larvae
  • Wg/Wnt1 and Erasp link ER stress to proapoptotic signaling in an autosomal dominant retinitis pigmentosa model
  • Disruption of the lipolysis pathway results in stem cell death through a sterile immunity-like pathway in adult Drosophila
  • Actin remodeling mediates ROS production and JNK activation to drive apoptosis-induced proliferation
  • E2F1 promotes, JNK and DIAP1 inhibit, and chromosomal position has little effect on radiation-induced Loss of Heterozygosity in Drosophila
  • Spoonbill positively regulates JNK signalling mediated apoptosis in Drosophila melanogaster
  • Bilateral JNK activation is a hallmark of interface surveillance and promotes elimination of aberrant cells
  • Mutation and apoptosis are well-coordinated for protecting against DNA damage-inducing toxicity in Drosophila
  • Epidermal growth factor receptor signaling protects epithelia from morphogenetic instability and tissue damage in Drosophila

    Mechanism of Apoptosis
  • 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 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
  • Screening of suppressors of bax-induced cell death identifies glycerophosphate oxidase-1 as a mediator of debcl-induced apoptosis in Drosophila
  • Akt1 and dCIZ1 promote cell survival from apoptotic caspase activation during regeneration and oncogenic overgrowth
  • Microtubule disassembly by caspases is an important rate-limiting step of cell extrusion
  • Necrosis-induced apoptosis promotes regeneration in Drosophila wing imaginal discs
  • Wingless mediated apoptosis: How cone cells direct the death of peripheral ommatidia in the developing Drosophila eye
  • 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
  • Overexpression of histone methyltransferase NSD in Drosophila induces apoptotic cell death via the Jun-N-terminal kinase pathway
  • Xrp1 is a transcription factor required for cell competition-driven elimination of loser cells
  • Simu-dependent clearance of dying cells regulates macrophage function and inflammation resolution
  • Natural genetic variation screen in Drosophila identifies Wnt signaling, mitochondrial metabolism, and redox homeostasis genes as modifiers of apoptosis
  • Xrn1/Pacman affects apoptosis and regulates expression of hid and reaper
  • Acheron/Larp6 Is a Survival Protein That Protects Skeletal Muscle From Programmed Cell Death During Development
  • Rab21 in enterocytes participates in intestinal epithelium maintenance
  • Toll-9 interacts with Toll-1 to mediate a feedback loop during apoptosis-induced proliferation in Drosophila
  • Dysfunction of lipid storage droplet-2 suppresses endoreplication and induces JNK pathway-mediated apoptotic cell death in Drosophila salivary glands
  • Non-autonomous cell death induced by the Draper phagocytosis receptor requires signaling through the JNK and SRC pathways
  • Apoptotic extracellular vesicle formation via local phosphatidylserine exposure drives efficient cell extrusion

    Apoptosis in neural development and neurons
  • Knockdown of the putative Lifeguard homologue CG3814 in neurons of Drosophila melanogaster
  • Identifying and monitoring neurons that undergo metamorphosis-regulated cell death (metamorphoptosis) by a neuron-specific caspase sensor (Casor) in Drosophila melanogaster
  • Combinatorial action of Grainyhead, Extradenticle and Notch in regulating Hox mediated apoptosis in Drosophila larval CNS
  • Culling less fit neurons protects against Amyloid-beta-induced brain damage and cognitive and motor decline
  • Two-factor specification of apoptosis: TGF-beta signaling acts cooperatively with ecdysone signaling to induce cell- and stage-specific apoptosis of larval neurons during metamorphosis in Drosophila melanogaster
  • Functional integration of "undead" neurons in the olfactory system
  • Three-tier regulation of cell number plasticity by neurotrophins and Tolls in Drosophila
  • Nutraceutical Strategy to Counteract Eye Neurodegeneration and Oxidative Stress in Drosophila melanogaster Fed with High-Sugar Diet
  • UQCRC1 engages cytochrome c for neuronal apoptotic cell death
  • The appearance of cytoplasmic cytochrome C precedes apoptosis during Drosophila salivary gland degradation
  • Decoupling developmental apoptosis and neuroblast proliferation in Drosophila
  • Drosophila FGFR/Htl signaling shapes embryonic glia to phagocytose apoptotic neurons
  • The dual role of heme oxygenase in regulating apoptosis in the nervous system of Drosophila melanogaster
  • Long-term sevoflurane exposure resulted in temporary rather than lasting cognitive impairment in Drosophila
  • Ensheathing glia promote increased lifespan and healthy brain aging
  • Non-muscle MYH10/myosin IIB recruits ESCRT-III to participate in autophagosome closure to maintain neuronal homeostasis

  • The lipolysis pathway sustains normal and transformed stem cells in adult Drosophila
  • Genetic analysis of dTSPO, an outer mitochondrial membrane protein, reveals its functions in apoptosis, longevity, and Ab42-induced neurodegeneration
  • Cardiac deficiency of single cytochrome oxidase assembly factor scox induces p53-dependent apoptosis in a Drosophila cardiomyopathy model
  • Ceramides and stress signalling intersect with autophagic defects in neurodegenerative Drosophila blue cheese (bchs) mutants
  • Selective endosomal microautophagy is starvation-inducible in Drosophila
  • Loss of Hsp67Bc leads to autolysosome enlargement in the Drosophila brain
  • Microenvironmental autophagy promotes tumour growth
  • Small chaperons and autophagy protected neurons from necrotic cell death
  • Generation and characterization of germline-specific autophagy and mitochondrial reactive oxygen species reporters in Drosophila
  • Lipid profiles of autophagic structures isolated from wild type and Atg2 mutant Drosophila
  • Yorkie drives supercompetition by non-autonomous induction of autophagy via bantam microRNA in Drosophila
  • DBT is a metabolic switch for maintenance of proteostasis under proteasomal impairment

    Autophagy, Physiology, Homeostasis and Development
  • An ancient defense system eliminates unfit cells from developing tissues during cell competition
  • Elimination of unfit cells maintains tissue health and prolongs lifespan
  • Cell competition is driven by autophagy
  • Fine-tuning autophagy maximises lifespan and is associated with changes in mitochondrial gene expression in Drosophila
  • A tissue- and temporal-specific autophagic switch controls Drosophila pre-metamorphic nutritional checkpoints
  • The metabolite alpha-ketoglutarate extends lifespan by inhibiting ATP synthase and TOR
  • Autophagy-dependent filopodial kinetics restrict synaptic partner choice during Drosophila brain wiring
  • Degradation of arouser by endosomal microautophagy is essential for adaptation to starvation in Drosophila
  • An autophagy-dependent tubular lysosomal network synchronizes degradative activity required for muscle remodeling
  • EGFR-dependent suppression of synaptic autophagy is required for neuronal circuit development
  • Lysosomal cystine mobilization shapes the response of TORC1 and tissue growth to fasting
  • ESCRT dysfunction compromises endoplasmic reticulum maturation and autophagosome biogenesis in Drosophila
  • Autophagy impairment and lifespan reduction caused by Atg1 RNAi or Atg18 RNAi expression in adult fruit flies (Drosophila melanogaster
  • Neuronal atg1 Coordinates Autophagy Induction and Physiological Adaptations to Balance mTORC1 Signalling
  • Polyploidy-associated autophagy promotes larval tracheal histolysis at Drosophila metamorphosis

    Autophagy, Immune Response, Aging and Disease
  • Transcriptional pausing controls a rapid antiviral innate immune response in Drosophila
  • Selective autophagy controls innate immune response through a TAK1/TAB2/SH3PX1 axis
  • Autophagy in neurodegeneration: two sides of the same coin
  • TFEB/Mitf links impaired nuclear import to autophagolysosomal dysfunction in C9-ALS
  • Inflammation-induced, STING-dependent autophagy restricts Zika virus infection in the Drosophila brain
  • Promoting basal levels of autophagy in the nervous system enhances longevity and oxidant resistance in adult Drosophila
  • 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
  • Autophagy within the mushroom body protects from synapse aging in a non-cell autonomous manner
  • Upregulation of the autophagy adaptor p62/SQSTM1 prolongs health and lifespan in middle-aged Drosophila
  • Activation of Nrf2 in Astrocytes Suppressed PD-Like Phenotypes via Antioxidant and Autophagy Pathways in Rat and Drosophila Models
  • Macros to Quantify Exosome Release and Autophagy at the Neuromuscular Junction of Drosophila Melanogaster
  • Progressive transcriptional changes in metabolic genes and altered fatbody homeostasis in Drosophila model of Huntington's disease
  • Autophagy is required for spermatogonial differentiation in the Drosophila testis
  • Proteostasis failure and mitochondrial dysfunction leads to aneuploidy-induced senescence
  • Circadian autophagy drives iTRF-mediated longevity
  • Inhibition of autophagy rescues muscle atrophy in a LGMDD2 Drosophila model
  • HEXA-018, a Novel Inducer of Autophagy, Rescues TDP-43 Toxicity in Neuronal Cells
  • Endophilin-B regulates autophagy during synapse development and neurodegeneration
  • A new model for fatty acid hydroxylase-associated neurodegeneration reveals mitochondrial and autophagy abnormalities
  • Autophagy controls Wolbachia infection upon bacterial damage and in aging Drosophila
  • Aging aggravates acetaminophen-induced acute liver injury and inflammation through inordinate C/EBPalpha-BMP9 crosstalk
  • Codon-optimized TDP-43 mediates neurodegeneration in a Drosophila model of ALS/FTLD
  • PI3P-dependent regulation of cell size and autophagy by phosphatidylinositol 5-phosphate 4-kinase
  • Atg2 Regulates Cellular and Humoral Immunity in Drosophila
  • Restoration of Sleep and Circadian Behavior by Autophagy Modulation in Huntington's Disease
  • Disrupted endoplasmic reticulum-mediated autophagosomal biogenesis in a Drosophila model of C9-ALS-FTD
  • Potent New Targets for Autophagy Enhancement to Delay Neuronal Ageing

    Mechanisms of Autophagy
  • Endophilin-B regulates autophagy during synapse development and neurodegeneration
  • Loss of Atg16 delays the alcohol-induced sedation response via regulation of Corazonin neuropeptide production in Drosophila
  • Human ZKSCAN3 and Drosophila M1BP are functionally homologous transcription factors in autophagy regulation
  • Defects of full-length dystrophin trigger retinal neuron damage and synapse alterations by disrupting functional autophagy
  • Drosophila NUAK functions with Starvin/BAG3 in autophagic protein turnover
  • Genes involved in autophagic cell death
  • Atg1-mediated myosin II activation regulates autophagosome formation during starvation-induced autophagy
  • Atg1 phosphorylation is activated by AMPK and indispensable for autophagy induction in insects
  • Atg9 Interacts with dTRAF2/TRAF6 to Regulate Oxidative Stress-Induced JNK Activation and Autophagy Induction
  • ARMS-NF-κB signaling regulates intracellular ROS to induce autophagy-associated cell death upon oxidative stress
  • Uba1 functions in Atg7- and Atg3-independent autophagy
  • 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
  • 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
  • Snazarus and its human ortholog SNX25 modulate autophagic flux>
  • 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
  • deubiquitinating enzyme UBPY is required for lysosomal biogenesis and productive autophagy in Drosophila
  • Drosophila Mitf regulates the V-ATPase and the lysosomal-autophagic pathway
  • 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
  • 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
  • The FUS gene is dual-coding with both proteins contributing to FUS-mediated toxicity
  • Multiple functions of the SNARE protein Snap29 in autophagy, endocytic, and exocytic trafficking during epithelial formation in Drosophila
  • Zonda is a novel early component of the autophagy pathway in Drosophila
  • Characterization of the Autophagy related gene-8a (Atg8a) promoter in Drosophila melanogaster
  • PtdIns4P exchange at endoplasmic reticulum-autolysosome contacts is essential for autophagy and neuronal homeostasis
  • A yeast two-hybrid screening identifies novel Atg8a interactors in Drosophila
  • Analysis of Drosophila Atg8 proteins reveals multiple lipidation-independent roles
  • Crosstalk between Dpp and Tor signaling coordinates autophagy-dependent midgut degradation
  • Hedgehog and Wingless signaling are not essential for autophagy-dependent cell death
  • Control of basal autophagy rate by vacuolar peduncle
  • Pleiotropic role of Drosophila phosphoribosyl pyrophosphate synthetase in autophagy and lysosome homeostasis
  • A conserved myotubularin-related phosphatase regulates autophagy by maintaining autophagic flux
  • Cp1/cathepsin L is required for autolysosomal clearance in Drosophila
  • PTPN9-mediated dephosphorylation of VTI1B promotes ATG16L1 precursor fusion and autophagosome formation
  • Autophagy inhibition rescues structural and functional defects caused by the loss of mitochondrial chaperone Hsc70-5 in Drosophila
  • Condition-dependent functional shift of two Drosophila Mtmr lipid phosphatases in autophagy control
  • Drosophila D-idua Reduction Mimics Mucopolysaccharidosis Type I Disease-Related Phenotypes
  • Mitochondrial aconitase 1 regulates age-related memory impairment via autophagy/mitophagy-mediated neural plasticity in middle-aged flies
  • Autophagy-mediated plasma membrane removal promotes the formation of epithelial syncytia
  • Loss of ubiquitinated protein autophagy is compensated by persistent cnc/NFE2L2/Nrf2 antioxidant responses
  • Atg6 promotes organismal health by suppression of cell stress and inflammation
  • Analyzing Starvation-Induced Autophagy in the Drosophila melanogaster Larval Fat Body
  • Glia-Neurons Cross-Talk Regulated Through Autophagy
  • The Drosophila effector caspase Dcp-1 regulates mitochondrial dynamics and autophagic flux via SesB
  • A coherent FOXO3-SNAI2 feed-forward loop in autophagy
  • Convergence of secretory, endosomal, and autophagic routes in trans-Golgi-associated lysosomes
  • A conserved interplay between FOXO and SNAI/snail in autophagy
  • ER-mitochondrial contact protein Miga regulates autophagy through Atg14 and Uvrag
  • A conserved STRIPAK complex is required for autophagy in muscle tissue
  • STING controls energy stress-induced autophagy and energy metabolism via STX17
  • Cyclin-G-associated kinase GAK/dAux regulates autophagy initiation via ULK1/Atg1 in glia

    Cell Deatb and Tumors
  • Tumor suppressor gene OSCP1/NOR1 regulates apoptosis, proliferation, differentiation, and ROS generation during eye development of Drosophila melanogaster
  • Non-apoptotic activation of Drosophila caspase-2/9 modulates JNK signaling, the tumor microenvironment, and growth of wound-like tumors
  • Apoptosis inhibition restrains primary malignant traits in different Drosophila cancer models
  • Lack of apoptosis leads to cellular senescence and tumorigenesis in Drosophila epithelial cells
  • Tumor suppressive autophagy in intestinal stem cells controls gut homeostasis
  • The autophagy-related gene Atg101 in Drosophila regulates both neuron and midgut homeostasis
  • V-ATPase controls tumor growth and autophagy in a Drosophila model of gliomagenesis
  • Autophagy induction in tumor surrounding cells promotes tumor growth in adult Drosophila intestines
  • Chromosomal instability-induced cell invasion through caspase-driven DNA damage

    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).

    A dual role of lola in Drosophila ovary development: regulating stem cell niche establishment and repressing apoptosis

    In Drosophila ovary, niche is composed of somatic cells, including terminal filament cells (TFCs), cap cells (CCs) and escort cells (ECs), which provide extrinsic signals to maintain stem cell renewal or initiate cell differentiation. Niche establishment begins in larval stages when terminal filaments (TFs) are formed, but the underlying mechanism for the development of TFs remains largely unknown. This study reports that transcription factor longitudinals lacking (Lola) is essential for ovary morphogenesis. Lola protein was expressed abundantly in TFCs and CCs, although also in other cells, and lola was required for the establishment of niche during larval stage. Importantly, it was found that knockdown expression of lola induced apoptosis in adult ovary, and that lola affected adult ovary morphogenesis by suppressing expression of Regulator of cullins 1b (Roc1b), an apoptosis-related gene that regulates caspase activation during spermatogenesis. These findings significantly expand understanding of the mechanisms controlling niche establishment and adult oogenesis in Drosophila (Zhao, 2022).

    Barrio, L., Gaspar, A. E., Muzzopappa, M., Ghosh, K., Romao, D., Clemente-Ruiz, M., Milan, M. (2023). Chromosomal instability-induced cell invasion through caspase-driven DNA damage. Curr Biol, 33(20):4446-4457.e4445 PubMed ID: 37751744

    Chromosomal instability-induced cell invasion through caspase-driven DNA damage

    Chromosomal instability (CIN), an increased rate of changes in chromosome structure and number, is observed in most sporadic human carcinomas with high metastatic activity. This study used a Drosophila epithelial model to show that DNA damage, as a result of the production of lagging chromosomes during mitosis and aneuploidy-induced replicative stress, contributes to CIN-induced invasiveness. A sub-lethal role of effector caspases in invasiveness was unraveled by enhancing CIN-induced DNA damage and identify the JAK/STAT signaling pathway as an activator of apoptotic caspases through transcriptional induction of pro-apoptotic genes. Evidence is provided that an autocrine feedforward amplification loop mediated by Upd3-a cytokine with homology to interleukin-6 and a ligand of the JAK/STAT signaling pathway-contributes to amplifying the activation levels of the apoptotic pathway in migrating cells, thus promoting CIN-induced invasiveness. This work sheds new light on the chromosome-signature-independent effects of CIN in metastasis (Barrio, 2023).

    Non-apoptotic caspase activation ensures the homeostasis of ovarian somatic stem cells

    Current evidence has associated caspase activation with the regulation of basic cellular functions without causing apoptosis. Malfunction of non-apoptotic caspase activities may contribute to specific neurological disorders, metabolic diseases, autoimmune conditions and cancers. However, understanding of non-apoptotic caspase functions remains limited. This study showed that non-apoptotic caspase activation prevents the intracellular accumulation of the Patched receptor in autophagosomes and the subsequent Patched-dependent induction of autophagy in Drosophila follicular stem cells. These events ultimately sustain Hedgehog signalling and the physiological properties of ovarian somatic stem cells and their progeny under moderate thermal stress. Importantly, the key findings are partially conserved in ovarian somatic cells of human origin. These observations attribute to caspases a pro-survival role under certain cellular conditions (Galasso. 2023).

    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)

    Akt1 and dCIZ1 promote cell survival from apoptotic caspase activation during regeneration and oncogenic overgrowth

    Apoptosis is an ancient and evolutionarily conserved cell suicide program. During apoptosis, executioner caspase enzyme activation has been considered a point of no return. However, emerging evidence suggests that some cells can survive caspase activation following exposure to apoptosis-inducing stresses, raising questions as to the physiological significance and underlying molecular mechanisms of this unexpected phenomenon. This study shows that, following severe tissue injury, Drosophila wing disc cells that survive executioner caspase activation contribute to tissue regeneration. Through RNAi screening, this study identified akt1 and a previously uncharacterized Drosophila gene CG8108, which is homologous to the human gene CIZ1, as essential for survival from the executioner caspase activation. It was also shown that cells expressing activated oncogenes experience apoptotic caspase activation, and that Akt1 and dCIZ1 are required for their survival and overgrowth. Thus, survival following executioner caspase activation is a normal tissue repair mechanism usurped to promote oncogene-driven overgrowth (Sun, 2020).

    Microtubule disassembly by caspases is an important rate-limiting step of cell extrusion

    The expulsion of dying epithelial cells requires well-orchestrated remodelling steps to maintain tissue sealing. This process, named cell extrusion, has been mostly analysed through the study of actomyosin regulation. Yet, the mechanistic relationship between caspase activation and cell extrusion is still poorly understood. Using the Drosophila pupal notum, a single layer epithelium where extrusions are caspase-dependent, this study showed that the initiation of cell extrusion and apical constriction are surprisingly not associated with the modulation of actomyosin concentration and dynamics. Instead, cell apical constriction is initiated by the disassembly of a medio-apical mesh of microtubules which is driven by effector caspases. Importantly, the depletion of microtubules is sufficient to bypass the requirement of caspases for cell extrusion, while microtubule stabilisation strongly impairs cell extrusion. This study shows that microtubules disassembly by caspases is a key rate-limiting step of extrusion, and outlines a more general function of microtubules in epithelial cell shape stabilisation (Villars, 2022).

    Non-apoptotic activation of Drosophila caspase-2/9 modulates JNK signaling, the tumor microenvironment, and growth of wound-like tumors

    Resistance to apoptosis due to caspase deregulation is considered one of the main hallmarks of cancer. However, the discovery of novel non-apoptotic caspase functions has revealed unknown intricacies about the interplay between these enzymes and tumor progression. To investigate this biological problem, this study capitalized on a Drosophila tumor model with human relevance based on the simultaneous overactivation of the EGFR and the JAK/STAT signaling pathways. The data indicate that widespread non-apoptotic activation of initiator caspases limits JNK signaling and facilitates cell fate commitment in these tumors, thus preventing the overgrowth and exacerbation of malignant features of transformed cells. Intriguingly, caspase activity also reduces the presence of macrophage-like cells with tumor-promoting properties in the tumor microenvironment. These findings assign tumor-suppressing activities to caspases independent of apoptosis, while providing molecular details to better understand the contribution of these enzymes to tumor progression (Xu, 2022). tumor

    Necrosis-induced apoptosis promotes regeneration in Drosophila wing imaginal discs

    Regeneration is a complex process that requires a coordinated genetic response to tissue loss. Signals from dying cells are crucial to this process and are best understood in the context of regeneration following programmed cell death, like apoptosis. Conversely, regeneration following unregulated forms of death, such as necrosis, have yet to be fully explored. This study has developed a method to investigate regeneration following necrosis using the Drosophila wing imaginal disc. Necrosis is shown to stimulate regeneration at an equivalent level to that of apoptosis-mediated cell death and activates a similar response at the wound edge involving localized JNK signaling. Unexpectedly, however, necrosis also results in significant apoptosis far from the site of ablation, which this study terms necrosis-induced apoptosis (NiA). This apoptosis occurs independent of changes at the wound edge and importantly does not rely on JNK signaling. Furthermore, it was found that blocking NiA limits proliferation and subsequently inhibits regeneration, suggesting that tissues damaged by necrosis can activate programmed cell death at a distance from the injury to promote regeneration (Klemm, 2021).

    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).

    ARMS-NF-κB signaling regulates intracellular ROS to induce autophagy-associated cell death upon oxidative stress

    Ankyrin repeat-rich membrane spanning (ARMS) plays roles in neural development, neuropathies, and tumor formation. Such pleiotropic function of ARMS is often attributed to diverse ARMS-interacting molecules in different cell context. However, it might be achieved by ARMS' effect on global biological mediator like reactive oxygen species (ROS). This study established ARMS-knockdown in melanoma cells (siARMS) and in Drosophila eyes (GMR>dARMS (RNAi)) and challenged them with H(2)O(2). Decreased ARMS in both systems compromises nuclear translocation of NF-κB and induces ROS, which in turn augments autophagy flux and confers susceptibility to H(2)O(2)-triggered autophagic cell death. Resuming NF-κB activity or reducing ROS by antioxidants in siARMS cells and GMR>dARMS (RNAi) fly decreases intracellular peroxides level concurrent with reduced autophagy and attenuated cell death. Conversely, blocking NF-κB activity in wild-type flies/melanoma enhances ROS and induces autophagy with cell death. This study has thus uncover intracellular ROS modulated by ARMS-NFκB signaling primes autophagy for autophagic cell death upon oxidative stress (Liao, 2023).

    Atg1 phosphorylation is activated by AMPK and indispensable for autophagy induction in insects

    Phosphorylation is a key post-translational modification in regulating autophagy in yeast and mammalians, yet it is not fully illustrated in invertebrates such as insects. ULK1/Atg1 is a functionally conserved serine/threonine protein kinase involved in autophagosome initiation. As a result of alternative splicing, Atg1 in the silkworm, Bombyx mori, is present as three mRNA isoforms, with BmAtg1c showing the highest expression levels. This study found that BmAtg1c mRNA expression, BmAtg1c protein expression and phosphorylation, and autophagy simultaneously peaked in the fat body during larval-pupal metamorphosis. Importantly, two BmAtg1c phosphorylation sites were identified at Ser269 and Ser270, which were activated by BmAMPK, the major energy-sensing kinase, upon stimulation with 20-hydroxyecdysone and starvation; additionally, these Atg1 phosphorylation sites are evolutionarily conserved in insects. The two BmAMPK-activated phosphorylation sites in BmAtg1c were found to be required for BmAMPK-induced autophagy. Moreover, the two corresponding DmAtg1 phosphorylation sites in the fruit fly, Drosophila melanogaster, are functionally conserved for autophagy induction. In conclusion, AMPK-activated Atg1 phosphorylation is indispensable for autophagy induction and evolutionarily conserved in insects, shedding light on how various groups of organisms differentially regulate ULK1/Atg1 phosphorylation for autophagy induction (Zhao, 2023).

    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).

    Hedgehog and Wingless signaling are not essential for autophagy-dependent cell death

    Autophagy-dependent cell death is a distinct mode of regulated cell death required in a context specific manner. One of the best validated genetic models of autophagy-dependent cell death is the removal of the Drosophila larval midgut during larval-pupal transition. Previous work has shown that down-regulation of growth signaling is essential for autophagy induction and larval midgut degradation. Sustained growth signaling through Ras and PI3K blocks autophagy and consequently inhibits midgut degradation. In addition, the morphogen Dpp plays an important role in regulating the correct timing of midgut degradation. This study explored the potential roles of Hh and Wg signaling in autophagy-dependent midgut cell death. Hh and Wg signaling are not involved in the regulation of autophagy-dependent cell death. However, surprisingly one key component of these pathways, the Drosophila Glycogen Synthase Kinase 3, Shaggy (Sgg), may regulate midgut cell size independent of Hh and Wg signaling (Xu, 2019).

    PTPN9-mediated dephosphorylation of VTI1B promotes ATG16L1 precursor fusion and autophagosome formation

    Macroautophagy/autophagy is an evolutionarily conserved intracellular pathway for the degradation of cytoplasmic materials. Under stress conditions, autophagy is upregulated and double-membrane autophagosomes are formed by the expansion of phagophores. The ATG16L1 (Drosophila homolog: ATG16) precursor fusion contributes to development of phagophore structures and is critical for the biogenesis of autophagosomes. This study discovered a novel role of the protein tyrosine phosphatase PTPN9 in the regulation of homotypic ATG16L1 vesicle fusion and early autophagosome formation. Depletion of PTPN9 and its Drosophila homolog Ptpmeg2 impaired autophagosome formation and autophagic flux. PTPN9 colocalized with ATG16L1 and was essential for homotypic fusion of ATG16L1(+) vesicles during starvation-induced autophagy. This study further identified the Q-SNARE VTI1B as a substrate target of PTPN9 phosphatase. Like PTPN9, the VTI1B nonphosphorylatable mutant but not the phosphomimetic mutant enhanced SNARE complex assembly and autophagic flux. These findings highlight the important role of PTPN9 in the regulation of ATG16L1(+) autophagosome precursor fusion and autophagosome biogenesis through modulation of VTI1B phosphorylation status (Chou, 2020).

    Autophagy inhibition rescues structural and functional defects caused by the loss of mitochondrial chaperone Hsc70-5 in Drosophila

    This study investigated in larval and adult Drosophila models whether loss of the mitochondrial chaperone Hsc70-5 is sufficient to cause pathological alterations commonly observed in Parkinson disease. At affected larval neuromuscular junctions, no effects on terminal size, bouton size or number, synapse size, or number were observed, suggesting that an early stage of pathogenesis was studied. At this stage, a loss of synaptic vesicle proteins and active zone components was observed, delayed synapse maturation, reduced evoked and spontaneous excitatory junctional potentials, increased synaptic fatigue, and cytoskeleton rearrangements. The adult model displayed ATP depletion, altered body posture, and susceptibility to heat-induced paralysis. Adult phenotypes could be suppressed by knockdown of dj-1β, Lrrk, DCTN2-p50, DCTN1-p150, Atg1, Atg101, Atg5, Atg7, and Atg12. The knockdown of components of the macroautophagy/autophagy machinery or overexpression of human HSPA9 broadly rescued larval and adult phenotypes, while disease-associated HSPA9 variants did not. Overexpression of Pink1 or promotion of autophagy exacerbated defects (Zhu, 2021).

    Autophagy induction in tumor surrounding cells promotes tumor growth in adult Drosophila intestines

    During tumorigenesis, tumor cells interact intimately with their surrounding cells (microenvironment) for their growth and progression. However, the roles of tumor microenvironment in tumor development and progression are not fully understood. Using an established benign tumor model in adult Drosophila intestines, this study found that non-cell autonomous autophagy (NAA) is induced in tumor surrounding neighbor cells. Tumor growth can be significantly suppressed by genetic ablation of autophagy induction in tumor neighboring cells, indicating that tumor neighboring cells act as tumor microenvironment to promote tumor growth. Autophagy in tumor neighboring cells is induced downstream of elevated ROS and activated JNK signaling in tumor cells. Interestingly, it was found that active transport of nutrients, such as amino acids, from tumor neighboring cells sustains tumor growth, and increasing nutrient availability could significantly restore tumor growth. Together, these data demonstrate that tumor cells take advantage of their surrounding normal neighbor cells as nutrient sources through NAA to meet their high metabolic demand for growth and progression. Thus this study provides insights into understanding of the mechanisms underlying the interaction between tumor cells and their microenvironment in tumor development (Zhao, 2021).

    Condition-dependent functional shift of two Drosophila Mtmr lipid phosphatases in autophagy control

    Myotubularin (MTM) and myotubularin-related (MTMR) lipid phosphatases catalyze the removal of a phosphate group from certain phosphatidylinositol derivatives. Because some of these substrates are required for macroautophagy/autophagy, during which unwanted cytoplasmic constituents are delivered into lysosomes for degradation, MTM and MTMRs function as important regulators of the autophagic process. Despite its physiological and medical significance, the specific role of individual MTMR paralogs in autophagy control remains largely unexplored. This study examined two Drosophila MTMRs, EDTP and Mtmr6, the fly orthologs of mammalian MTMR14 and MTMR6 to MTMR8, respectively; these enzymes were found to affect the autophagic process in a complex, condition-dependent way. EDTP inhibited basal autophagy, but did not influence stress-induced autophagy. In contrast, Mtmr6 promoted the process under nutrient-rich settings, but effectively blocked its hyperactivation in response to stress. Thus, Mtmr6 is the first identified MTMR phosphatase with dual, antagonistic roles in the regulation of autophagy, and shows conditional antagonism/synergism with EDTP in modulating autophagic breakdown. These results provide a deeper insight into the adjustment of autophagy (Manzeger, 2021).

    Drosophila D-idua Reduction Mimics Mucopolysaccharidosis Type I Disease-Related Phenotypes

    Deficit of the IDUA (α-L-iduronidase) enzyme causes the lysosomal storage disorder mucopolysaccharidosis type I (MPS I), a rare pediatric neurometabolic disease, due to pathological variants in the IDUA gene and is characterized by the accumulation of the undegraded mucopolysaccharides heparan sulfate and dermatan sulfate into lysosomes, with secondary cellular consequences that are still mostly unclarified. This paper reports a new fruit fly RNAi-mediated knockdown model of a IDUA homolog (D-idua) displaying a phenotype mimicking some typical molecular features of Lysosomal Storage Disorders (LSD). This study showed that D-idua is a vital gene in Drosophila and that ubiquitous reduction of its expression leads to lethality during the pupal stage, when the precise degradation/synthesis of macromolecules, together with a functional autophagic pathway, are indispensable for the correct development to the adult stage. Tissue-specific analysis of the D-idua model showed an increase in the number and size of lysosomes in the brain and muscle. Moreover, the incorrect acidification of lysosomes led to dysfunctional lysosome-autophagosome fusion and the consequent block of autophagy flux. A concomitant metabolic drift of glycolysis and lipogenesis pathways was observed. After starvation, D-idua larvae showed a quite complete rescue of both autophagy/lysosome phenotypes and metabolic alterations. Metabolism and autophagy are strictly interconnected vital processes that contribute to maintain homeostatic control of energy balance, and little is known about this regulation in LSDs. These results provide new starting points for future investigations on the disease's pathogenic mechanisms and possible pharmacological manipulations (De Filippis, 2021).

    Mitochondrial aconitase 1 regulates age-related memory impairment via autophagy/mitophagy-mediated neural plasticity in middle-aged flies
    Age-related memory impairment (AMI) occurs in many species, including humans. The underlying mechanisms are not fully understood. In wild-type Drosophila (w1118), AMI appears in the form of a decrease in learning (3-min memory) from middle age (30 days after eclosion [DAE]). in vivo, DNA microarray, and behavioral screen studies were performed to identify genes controlling both lifespan and AMI and mitochondrial Acon1 (mAcon1) was selected. mAcon1 expression in the head of w(1118) decreased with age. Neuronal overexpression of mAcon1 extended its lifespan and improved AMI. Neuronal or mushroom body expression of mAcon1 regulated the learning of young (10 DAE) and middle-aged flies. Interestingly, acetyl-CoA and citrate levels increased in the heads of middle-aged and neuronal mAcon1 knockdown flies. Acetyl-CoA, as a cellular energy sensor, is related to autophagy. Autophagy activity and efficacy determined by the positive and negative changes in the expression levels of Atg8a-II and p62 were proportional to the expression level of mAcon1. Levels of the presynaptic active zone scaffold protein Bruchpilot were inversely proportional to neuronal mAcon1 levels in the whole brain. Furthermore, mAcon1 overexpression in Kenyon cells induced mitophagy labeled with mt-Keima and improved learning ability. Both processes were blocked by pink1 knockdown. Taken together, these results imply that the regulation of learning and AMI by mAcon1 occurs via autophagy/mitophagy-mediated neural plasticity (Choy, 2021).

    Autophagy-mediated plasma membrane removal promotes the formation of epithelial syncytia

    Epithelial wound healing in Drosophila involves the formation of multinucleate cells surrounding the wound. This study shows that autophagy, a cellular degradation process often deployed in stress responses, is required for the formation of a multinucleated syncytium during wound healing, and that autophagosomes that appear near the wound edge acquire plasma membrane markers. In addition, uncontrolled autophagy in the unwounded epidermis leads to the degradation of endo-membranes and the lateral plasma membrane, while apical and basal membranes and epithelial barrier function remain intact. Proper functioning of TORC1 is needed to prevent destruction of the larval epidermis by autophagy, in a process that depends on phagophore initiation and expansion but does not require autophagosomes fusion with lysosomes. Autophagy induction can also affect other sub-cellular membranes, as shown by its suppression of experimentally induced laminopathy-like nuclear defects. These findings reveal a function for TORC1-mediated regulation of autophagy in maintaining membrane integrity and homeostasis in the epidermis and during wound healing (Kakanj, 2022).

    Loss of ubiquitinated protein autophagy is compensated by persistent cnc/NFE2L2/Nrf2 antioxidant responses

    SQSTM1/p62-type selective macroautophagy/autophagy receptors cross-link poly-ubiquitinated cargo and autophagosomal LC3/Atg8 proteins to deliver them for lysosomal degradation. Consequently, loss of autophagy leads to accumulation of polyubiquitinated protein aggregates that are also frequently seen in various human diseases, but their physiological relevance is incompletely understood. Using a genetically non-redundant Drosophila model, this study shows that specific disruption of ubiquitinated protein autophagy and concomitant formation of polyubiquitinated aggregates has hardly any effect on bulk autophagy, proteasome activity and fly healthspan. Accumulation of ref(2)P/SQSTM1 due to a mutation that disrupts its binding to Atg8a results in the co-sequestering of Keap1 and thus activates the cnc/NFE2L2/Nrf2 antioxidant pathway. These mutant flies have increased tolerance to oxidative stress and reduced levels of aging-associated mitochondrial superoxide. Interestingly, ubiquitin overexpression in ref(2)P point mutants prevents the formation of large aggregates and restores the cargo recognition ability of ref(2)P, although it does not prevent the activation of antioxidant responses. Taken together, potential detrimental effects of impaired ubiquitinated protein autophagy are compensated by the aggregation-induced antioxidant response (Bhattacharjee, 2022).

    Atg6 promotes organismal health by suppression of cell stress and inflammation

    Autophagy targets cytoplasmic materials for degradation, and influences cell health. Alterations in Atg6/Beclin-1, a key regulator of autophagy, are associated with multiple diseases. While the role of Atg6 in autophagy regulation is heavily studied, the role of Atg6 in organism health and disease progression remains poorly understood. This study discovered that loss of Atg6 in Drosophila results in various alterations to stress, metabolic and immune signaling pathways. The increased levels of circulating blood cells and tumor-like masses in atg6 mutants vary depending on tissue-specific function of Atg6, with contributions from intestine and hematopoietic cells. These phenotypes are suppressed by decreased function of macrophage and inflammatory response receptors crq and drpr. Thus, these findings provide a basis for understanding how Atg6 systemically regulates cell health within multiple organs, and highlight the importance of Atg6 in inflammation to organismal health (Shen, 2022).

    Atg6/Beclin-1 has been implicated in the regulation of autophagy, endocytosis, apoptosis, and other cell processes. These diverse cellular roles suggest how Atg6/Beclin-1 loss may lead to health detriments and disease development. In mice, Beclin-1 loss enhanced tumorigenesis and invasive potential. Breast cancer cells deficient in Beclin-1 demonstrate enhanced tumorgenicity, potentially through modifications to cell membrane proteins like E-Cadherin. Like these mammalian models, Drosophila larvae lacking Atg6 possess cells with invasive properties in the form of blood cell masses. This study demonstrated the complex and tissue-specific roles of Atg6 by showing Atg6 rescue specifically in either blood or intestine cells differentially modulates atg6 deficiency phenotypes. Importantly, it was established that the macrophage receptors Crq and Drpr contribute to atg6 mutant blood cell tumor-like aggregation, providing strong evidence of Atg6 function as a suppressor of inflammation (Shen, 2022).

    Studies of RNA levels in atg6 mutants revealed significant changes in multiple pathways. Stress response genes, including those in the Turandot and glutathione S-transferase family, were strongly upregulated with loss of atg6. These changes may reflect an increase in cellular stress, consistent with the metabolic roles of Atg6. Consistent with this possibility, a substantial difference in metabolite profiles were detected between atg6 mutants and controls in multiple tissue types. An increase was detected in specific metabolites in the atg6 mutant intestine and hemolymph, including phosphoserine and phosphoethanolamine. Interestingly, modulators of phosphoserine are known to affect cancer prognosis. In addition, previous work demonstrated that phosphoethanolamine accumulation protected breast cancer cells from glutamine deprivation and enhanced tumorigenicity. RNAs of genes associated with ribosomes were also increased with loss of Atg6, and increased ribosome biogenesis is a hallmark of many cancer types. Metabolic changes may provide an enhanced environment for tumor cells to establish and spread, which may explain why atg6 mutants are so prone to developing invasive blood cell masses. It is tempting to speculate if haploinsufficient beclin-1 mice, which also share a predisposition for developing spontaneous tumors, have a similar metabolic profile that promotes tumorous growth. Future analyses of the metabolic changes observed in atg6 loss-of-function mutant Drosophila in haplo-deficient beclin-1 mice will help address this question (Shen, 2022).

    Alterations in stress response pathways and imbalances in amino acid content can adversely affect the health of the cell, potentially explaining the increased ROS in atg6 loss-of-function mutant intestine cells. It is certainly possible that with loss of Atg6 function, cells are unable to clear unstable cargo and waste through autophagy, leading to increased ROS. The mitophagy deficiency in intestine cells with decreased Atg6 function is consistent with this hypothesis, as a failure to clear dysfunctional mitochondria could result in increased mitochondrial ROS. However, it is unknown if this principle applies to non-mitochondrial autophagic cargoes. Furthermore, the possibility of a non-autophagy-related role of Atg6 in ROS production cannot be ruled out. Thus, these findings open new directions for how Atg6/Beclin-1 may function in tissue-specific manners to promote survival and stress-response during development, as well as suppress tumor formation (Shen, 2022).

    Alterations in stress response markers, including GSTD1-GFP and TRE-GFP, in atg6 mutant intestine cells also support a role for Atg6 in suppression of stress. Furthermore, the upregulation of BomS1, BomS2, BomS3, Dso1, as well as genes involved in metabolism in atg6 mutants may be consistent with the role of Atg6 in inflammation and immunity. Loss of Atg6 could also affect nutrient-sensing pathways because of the established role of autophagy in catabolism, which could explain decreased activation of AKT and increased levels of phosphorylated AMPK. AKT normally inhibits Atg6 activity in response to growth factors. The nutrient-deprived and sepsis-like characteristics of Atg6 loss-of-function mutants are counter-intuitive with AKT activity. Likewise, activation of AMPK serves to upregulate Atg6 activity, which would be expected to be increased if Atg6 activity is missing and the animal is deprived of nutrients. Further investigation will determine if these phenotypes are due to either direct roles of Atg6 or if they reflect more global and indirect effects of Atg6 deficiency (Shen, 2022).

    Previous work suggests that AKT activates SREBP. Therefore, it is interesting that SREBP activity is increased in atg6 mutants when AKT activity is decreased. It is possible that loss of Atg6 uncouples this relationship by alteration of lipid content. While speculative, decreased lipid droplets in cells may stimulate SREBP activity, which through a negative feedback loop could inhibit AKT activity. Further studies, including investigation of the role of mTOR in these pathways, are necessary to elucidate the relationship between SREBP and AKT in the absence of Atg6 (Shen, 2022).

    Characterization of the blood cell masses in atg6 mutant larvae demonstrates that Atg6-deficient blood cells display invasive phenotypes, including MMP1 positivity and disruption of intestine smooth muscle. Furthermore, atg6 mutant phenotypes, including developmental arrest, blood cell aggregation, lymph gland enlargement, and increased circulating blood cell numbers, were suppressed to varying degrees by expression of Atg6 in either intestine or blood cells. However, rescue expression in some tissue subsets failed to suppress mutant lethality or blood cell phenotypes, supporting a hypothesis that altered Atg6 function in a subset of tissues could drive tumorigenesis and disrupt systemic homeostasis. Future studies should evaluate how Atg6 loss in one tissue affects metabolites in tissues that retain Atg6 function (Shen, 2022).

    Significantly, the blood cell immune receptors Crq and Drpr contributed to blood cell aggregation in atg6 mutants. It remains unclear if similar inflammatory mechanisms account for both the blood cell differences and tumors in Beclin-1 deficient mice. Mmp1 plays a role in cancer invasiveness, likely as a collagenase that facilitates tumor cell invasion through the basement membrane . Although speculative, increased Mmp1 activity resulting from loss of Atg6/Beclin-1 and subsequent dysregulation of Crq and/or Drpr may explain the increase in invasive cell types in both Drosophila and mice. However, it cannot be excluded that increased Mmp1 activity is simply an indirect byproduct of atg6 loss (Shen, 2022).

    Interestingly, Beclin-1 provides a protective role against sepsis (Sun, 2018). When compared to control animals, atg6 mutants display several molecular and metabolic hallmarks of sepsis. Upregulation of AICAR and AMP, as well as subsequent increase in pAMPK, is consistent with an increased response to septic shock. As in human patients with sepsis, atg6 mutants display an acute inflammatory response, immune cell activation, and increased oxidative stress as evidenced by JNK activation, upregulation of immune response genes, and increased oxidative stress. Likewise, MMP1 activation is believed to contribute to sepsis development. Taken together, these findings are consistent with a model whereby atg6 deficiency either results in increased susceptibility to sepsis or upregulates a sepsis-like response. These findings could explain the increased number of circulating blood cells, as increased circulating immune cells are characteristic of sepsis. In addition, the fact that Atg6 expression specifically in immune cell lineages rescues multiple mutant phenotypes suggests that Atg6 regulates this process, at least in part, through its role in the immune system. However, the possibility that loss of Atg6 contributes to this phenotype by enhancing blood cell proliferation through upregulation of cell proliferation regulators. Additional studies of Atg6 function in sepsis development, along with other diseases including cancer, will be required to better understand how Beclin-1 suppresses these human conditions (Shen, 2022).

    Analyzing Starvation-Induced Autophagy in the Drosophila melanogaster Larval Fat Body

    Autophagy is a cellular self-digestion process. It delivers cargo to the lysosomes for degradation in response to various stresses, including starvation. The malfunction of autophagy is associated with aging and multiple human diseases. The autophagy machinery is highly conserved-from yeast to humans. The larval fat body of Drosophila melanogaster, an analog for vertebrate liver and adipose tissue, provides a unique model for monitoring autophagy in vivo. Autophagy can be easily induced by nutrient starvation in the larval fat body. Most autophagy-related genes are conserved in Drosophila. Many transgenic fly strains expressing tagged autophagy markers have been developed, which facilitates the monitoring of different steps in the autophagy process. The clonal analysis enables a close comparison of autophagy markers in cells with different genotypes in the same piece of tissue. The current protocol details procedures for (1) generating somatic clones in the larval fat body, (2) inducing autophagy via amino acid starvation, and (3) dissecting the larval fat body, aiming to create a model for analyzing differences in autophagy using an autophagosome marker (GFP-Atg8a) and clonal analysis (Shi, 2022).

    Glia-Neurons Cross-Talk Regulated Through Autophagy

    Autophagy is a self-degradative process which plays a role in removing misfolded or aggregated proteins, clearing damaged organelles, but also in changes of cell membrane size and shape. The aim of this phenomenon is to deliver cytoplasmic cargo to the lysosome through the intermediary of a double membrane-bound vesicle (autophagosome), that fuses with a lysosome to form autolysosome, where cargo is degraded by proteases. Products of degradation are transported back to the cytoplasm, where they can be re-used. This study showed that autophagy is important for proper functioning of the glia and that it is involved in the regulation of circadian structural changes in processes of the pacemaker neurons. This effect is mainly observed in astrocyte-like glia, which play a role of peripheral circadian oscillators in the Drosophila brain (Damulewicz, 2022).

    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).

    Fine-tuning autophagy maximises lifespan and is associated with changes in mitochondrial gene expression in Drosophila

    Increased cellular degradation by autophagy is a feature of many interventions that delay ageing. This paper reports that increased autophagy is necessary for reduced insulin-like signalling (IIS) to extend lifespan in Drosophila and is sufficient on its own to increase lifespan. It was first established that the well-characterised lifespan extension associated with deletion of the insulin receptor substrate chico was completely abrogated by downregulation of the essential autophagy gene Atg5. Next autophagy was directly induced by over-expressing the major autophagy kinase Atg1; a mild increase in autophagy extended lifespan. Interestingly, strong Atg1 up-regulation was detrimental to lifespan. Transcriptomic and metabolomic approaches identified specific signatures mediated by varying levels of autophagy in flies. Transcriptional upregulation of mitochondrial-related genes was the signature most specifically associated with mild Atg1 upregulation and extended lifespan, whereas short-lived flies, possessing strong Atg1 overexpression, showed reduced mitochondrial metabolism and up-regulated immune system pathways. Increased proteasomal activity and reduced triacylglycerol levels were features shared by both moderate and high Atg1 overexpression conditions. These contrasting effects of autophagy on ageing and differential metabolic profiles highlight the importance of fine-tuning autophagy levels to achieve optimal healthspan and disease prevention (Bjedov, 2020).

    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).

    Selective autophagy controls innate immune response through a TAK1/TAB2/SH3PX1 axis

    Selective autophagy is a catabolic route that turns over specific cellular material for degradation by lysosomes, and whose role in the regulation of innate immunity is largely unexplored. This study shows that the apical kinase of the Drosophila immune deficiency (IMD) pathway Tak1, as well as its co-activator Tab2, are both selective autophagy substrates that interact with the autophagy protein Atg8a. A role is presented for the Atg8a-interacting protein Sh3px1 in the downregulation of the IMD pathway, by facilitating targeting of the Tak1/Tab2 complex to the autophagy platform through its interaction with Tab2. These findings show the Tak1/Tab2/Sh3px1 interactions with Atg8a mediate the removal of the Tak1/Tab2 signaling complex by selective autophagy. This in turn prevents constitutive activation of the IMD pathway in Drosophila. This study provides mechanistic insight on the regulation of innate immune responses by selective autophagy (Tsapras, 2022a).

    Analysis of Drosophila Atg8 proteins reveals multiple lipidation-independent roles

    Yeast Atg8 and its homologs are involved in autophagosome biogenesis in all eukaryotes. These are the most widely used markers for autophagy thanks to the association of their lipidated forms with autophagic membranes. The Atg8 protein family expanded in animals and plants, with most Drosophila species having two Atg8 homologs. This report used clear-cut genetic analysis in Drosophila melanogaster to show that lipidated Atg8a is required for autophagy, while its non-lipidated form is essential for developmentally programmed larval midgut elimination and viability. In contrast, expression of Atg8b is restricted to the male germline and its loss causes male sterility without affecting autophagy. High expression of non-lipidated Atg8b in the male germline was found to be required for fertility. Consistent with these non-canonical functions of Atg8 proteins, loss of Atg genes required for Atg8 lipidation lead to autophagy defects but do not cause lethality or male sterility (Jipa, 2021).

    At first, the evolution of Atg8 genes was reconstructed and visualized in a species tree, showing the number and type of Atg8 proteins in selected insect and closely related arthropod species. This analysis revealed the presence of at least one Atg8 homolog in each species, as expected. An Atg8 paralog appeared early on that has been lost in many species later on, including those that belong to the family of Drosophilidae. Interestingly, all Drosophila species have another Atg8 paralog. In Drosophila melanogaster, these proteins are called Atg8a and Atg8b, respectively. Of note, it is the homologs of Atg8a that are found in all species that were analyzed, and the Drosophilidae-specific Atg8b gene probably arose in a retrotransposition event because it lacks introns (Jipa, 2021).

    The amino acid sequences of insect Atg8a and Drosophilidae-specific Atg8b proteins are very similar and are closer to the human GABARAP subfamily than to MAP1LC3 proteins. In order to gain insight into the functions of Atg8a and Atg8b, genetic null alleles were created for both in Drosophila melanogaster. Previous functional studies of Atg8a relied on two viable alleles: a P-element insertion into the protein-coding sequence in the first exon of the main Atg8a isoform (Atg8aKG07569), and a molecularly defined deletion extending from this insertion site into the promoter region (Atg8ad4), which was generated by imprecise excision of this P element. Importantly, high-throughput expression analyses identified the presence of two alternative Atg8a promoters, which can produce another two Atg8a isoforms that differ in their N-termini from the main isoform. Thus, these two previously described Atg8a mutations are likely not genetic null alleles (Jipa, 2021).

    Advantage was taken of a Minos transposable element insertion (Atg8aMI13726) in the first intron of Atg8a that is shared by all three alternatively spliced isoforms by inserting a new protein-coding exon called Trojan-Gal4 into the Minos element that traps all isoforms. This new insertion generated fusions between the very N-terminal parts of all Atg8a protein isoforms and a self-cleaving T2A polypeptide coupled to a yeast Gal4 transcription factor (a widely used genetic tool for driving the expression of genes containing a UAS promoter in Drosophila). The new allele (Atg8aTro-Gal4) thus prevented the expression of all Atg8a protein isoforms and was likely a genetic null. This was supported by the late pupal lethality of Atg8aTro-Gal4 mutant animals, and also by the accumulation of ref(2)P/p62 (refractory to sigma P; a receptor and selective autophagic cargo in Drosophila) in western blots. Of note, low ref(2)P levels were restored by introducing a 3xmCherry-Atg8a transgene driven by its genomic promoter in both Atg8aKG07569 and Atg8aTro-Gal4 homozygous mutant larvae (Jipa, 2021).

    The glycine residue located near the C-terminal end of Atg8 homologs is required for their lipidation. This motivated the generation of a non-lipidatable mutant form that could be used for further functional analyses. The CRISPR-Cas9 system was used to introduce a point mutation into the endogenous Atg8a locus, which mutated the 116th glycine into a stop codon. Animals carrying this Atg8aG116* mutation indeed showed no sign of Atg8a lipidation but were viable and fertile with no overall morphological alterations (Jipa, 2021).

    Since no mutants have been previously described for Atg8b, CRISPR/Cas9 gene targeting was used to delete the whole protein-coding sequence of this gene. Unlike the Atg8aTro-Gal4 mutation that appeared to eliminate the expression of Atg8a based on using a commercial anti-pan-GABARAP antibody (previously verified for recognizing Drosophila Atg8 in western blots of larval lysates, the Atg8b16 gene deletion had no obvious effect on Atg8a protein levels in such samples. This was in line with high-throughput expression analyses indicating that Atg8b is a testis-specific protein. Adult testis samples were also analyzed by western blotting, which indeed detected Atg8b expression: a clear band corresponding to this protein was present in controls as well as viable Atg8ad4 and Atg55CC5 mutant animals, with these Atg8a and Atg5 mutants showing no expression or lack of lipidation of Atg8a, respectively (Jipa, 2021).

    Next, autophagic activity was analyzed in the new Atg8a and Atg8b alleles using confocal microscopy. Immunofluorescent analyses using anti-GABARAP/Atg8 detected no signal in Atg8aTro-Gal4 mutant cell clones (marked by the lack of GFP) in mosaic fat tissue of starved larvae, while a faint cloud-like signal was seen in Atg8aG116** mutant cells (Figure 2B), likely representing nonlipidated Atg8a-I. LysoTracker Red is commonly used to stain acidic autolysosomes in Drosophila fat cells. Both Atg8aTro-Gal4 and Atg8aG116** cell clones showed impaired starvation-induced punctate LysoTracker staining compared to neighboring control cells. Lastly, aggregates of the selective autophagy cargo GFP-ref(2)P accumulated in both Atg8aTro-Gal4 and Atg8aG116** mutant cell clones (marked by lack of RFP). These data altogether indicated that autophagy was blocked in cells homozygous for either of these Atg8a alleles, as expected. In contrast, punctate LysoTracker staining was indistinguishable in homozygous mutant Atg8b16 cell clones (marked by GFP) from neighboring control fat cells of starved larvae, and there was no difference in the levels of endogenous ref(2)P between Atg8b cells (marked by lack of GFP expression) and controls. Thus, Atg8b was dispensable for autophagy in fat cells, in line with its testis-specific expression (Jipa, 2021).

    Elimination of the larval midgut epithelium during metamorphosis is considered to involve a form of developmentally programmed autophagic cell death/cell shrinkage. Surprisingly, proteins required for Atg8 lipidation turned out to be dispensable for this process, even though it requires most other Atg genes including Atg8a itself. These previous studies left the question open whether this phenomenon is due to an alternative pathway of Atg8a lipidation or represents a lipidation-independent role of Atg8a in larval midgut elimination. To answer this question, gastric ceca regression that largely takes place in the first 4 h of metamorphosis, as this process is commonly used to monitor larval midgut elimination. The four gastric ceca are out-growths of the anterior larval midgut that were clearly visible at the time of puparium formation but largely disappeared by 4 h later. This process was strongly impaired in animals mutant for Atg8aTro-Gal4, in line with previous studies of Atg8a requirement. However, gastric ceca regression happened normally in animals unable to lipidate Atg8a, pointing to a lipidation-independent role of Atg8a in this process. Lastly, the lack of Atg8b had no effect on developmental gastric ceca elimination either, in line with the testis-specific expression of this gene. Interestingly, impaired gastric ceca retraction of the Atg8aTro-Gal4 mutant was rescued by endogenous Atg8a promoter-driven expression of either Atg8a or Atg8b, indicating that both Atg8 homologs had the potential to promote gastric ceca regression if present in this tissue. Similar phenotypes were also obvious in pupae: animals mutant for either Atg8aG116** or Atg8b16 were viable and morphologically indistinguishable from controls, whereas Atg8aTro-Gal4 null mutants were much smaller with defects in the eversion of anterior spiracles (respiratory openings), and all died before eclosion (Jipa, 2021).

    To gain further insight into the function of Atg8a and Atg8b, their expression patterns were examined. The previously described 3xmCherry-Atg8a reporter that is driven by the endogenous Atg8a promoter and contains all Atg8a exons and introns showed universal expression in all tissues, in line with its important role in autophagy in all cells. A 3xeGFP-Atg8b reporter was generated driven by the endogenous Atg8b promoter. The expression of this transgene was only detected in the developing testis in larvae. The expression of these proteins was analyzed in the adult testis. Transgenic 3xmCherry-Atg8a expression was detected in both the germline and somatic cells of the testis. In contrast, transgenic 3xGFP-Atg8b was highly expressed in the germline and was absent from somatic cells. During spermiogenesis, both Atg8a and Atg8b displayed punctate distribution with a diffuse background in early cysts. Interestingly, while Atg8a expression strongly decreased in post-meiotic stages, the high-level expression of Atg8b was maintained in these elongated cysts, and Atg8b was clearly associated with the tail region of spermatids (Jipa, 2021).

    These observations prompted fertility tests with males mutant for different Atg genes. Crossing these Atg mutants to control females revealed that only 'Atg8b males were sterile, unlike Atg5, Atg7, Atg16 (note that the corresponding proteins are necessary for Atg8 lipidation), Atg101 (encoding an Atg1 kinase subunit) and Atg9 (encoding a transmembrane protein) null mutant males. While a subset of Atg8aG116** mutant males were also sterile; this was likely due to defects in wing unfolding that were observed at a low penetrance, as most males homozygous for this mutation managed to produce offspring. Although autophagy is important to prevent a decline in fertility over time owing to the need for long-term maintenance of stem cells, all of the viable Atg mutants that were tested can be maintained as homozygous stocks with the exception of male-sterile Atg8b nulls and female sterile Atg9 nulls (Jipa, 2021).

    To understand the reason for the sterility of 'Atg8b males, microscopic analysis was conducted on dissected testis samples, looking for aberrations in characteristic developmental stages. No abnormalities were detected in the early developmental stages, as the 16-cell cysts containing primary spermatocytes were formed and meioses seemed to proceed normally in the mutant. After meiosis the spermatids start to elongate, giving rise to elongated cysts in both control and mutant males, therefore this process was not affected in Atg8b null males. After elongation, the next developmental step is individualization. During this, the individualization complex (which consists of actin-rich, cone-shaped cytoskeletal structures) forms at the apical end of the cyst and starts its migration to the basal end. The majority of cytosolic components are degraded and expelled to the waste bag, and at the end of the process, the individual sperm forms. For spermatid individualization, directed protein degradation by proteasomes and non-apoptotic caspase activity are essential. Caspase activity was visualized with anti-active Drice antibody and migrating actin cones with fluorophore-conjugated phalloidin. The individualization process was found to proceed normally in Atg8b mutants, and the morphology of the individualization complex was similar to the controls, and the non-apoptotic caspase cascade was active in the forming waste bags. After individualization, the next step is the coiling and transfer of the mature sperm to the seminal vesicle. This step was studied by visualizing mature polyglycylated axonemal tubulins with the AXO49 antibody. This analysis indicated that mature sperm formed in the 'Atg8b, but its proper transfer was defective. In line with this, the enlargement of the proximal part of the testis due to accumulation of sperm was obvious in 'Atg8b males. To test if the sperms reaching the seminal vesicle were transferred to the females or not, they were marked with dj-GFP that properly labels both control and 'Atg8b sperm cells. After mating these males to control females, the female sperm storage organs were analyaed. These experiments revealed that Atg8b sperm cells failed to reach the seminal receptacle and spermatheca of mated control females. This result was probably due to the low motility of mutant sperm cells. Since transmission electron microscopy showed normal spermatid ultrastructure in the developing cysts in Atg8b mutants, as the axoneme and the two mitochondrial derivatives formed normally, these suggested that Atg8b did not function as a structural protein in spermiogenesis. Taken together, this study pinpointed the role of Atg8b at the late stages of post-meiotic spermatid development and motility (Jipa, 2021).

    This analysis of multiple viable Atg mutants suggested that it was likely not autophagy that causes infertility in Drosophila males. The importance of potential Atg8b lipidation was tested. Transgenic expression of full-length Atg8b driven by its endogenous promoter readily rescued the male sterility of Atg8b null mutants, similar to an Atg8bΔG-Flag transgene lacking the glycine residue in the C-terminal part of the protein that would be critical for lipidation. Autophagy was assessed in the testis by counting the number of GFP-ref(2)P dots in testis samples. Similar to fat cell data, GFP-ref(2)P aggregates accumulated in Atg8a mutants compared to control and Atg8bG116* mutant males. These results, together with Atg8b always appearing as a single band in western blots and that proteins involved in Atg8 lipidation and autophagy were dispensable for male fertility, strongly supported a lipidation- and autophagy-independent role for Atg8b in this process. Interestingly, while the amino acid sequence of Atg8b orthologs was almost identical among various Drosophila species, in Drosophila obscura, persimilis, miranda, guanche, and pseudoobscura (all belonging to the obscura group) a C-terminal truncation of a few amino acids led to complete loss of the glycine that would be essential for lipidation. further supporting that Atg8b lipid conjugation was not critical for its testis function. One possibility is a potential microtubule-associated role that is also in line with the localization of Atg8b in the tails of elongating spermatids. Interestingly, the Atg8b phenotype manifested after individualization, where the sperm cells already had minimal cytosol, and the axoneme was surrounded by an ER-derived double membrane. Since all these specialized structures form normally in the absence of Atg8b based on ultrastructural analysis, the exact role of Atg8b in the testis remained unclear (Jipa, 2021).

    The last question that was addressed was the following: what is so special about Atg8b? Is it required for male fertility because of its high expression in the male germline so that the lower expression of Atg8a cannot compensate for its loss, or did the function of this protein diverge from that of Atg8a? To this end, an Atg8a transgene driven was generated by the testis-specific promoter of Atg8b. Expression of this construct perfectly restored male fertility in Atg8b mutants, suggesting that a high level of either non-lipidated Atg8 protein is sufficient for male fertility in Drosophila. The testis-specific function of Atg8b is consistent with the common generation of new autosomal retrogenes with testis-specific expression (including Atg8b) from X-linked genes (including Atg8a), a process that is likely driven by X chromosome inactivation during late spermatogenesis in both Drosophila and mammals. These data pointed to a potential general importance of Atg8 family proteins in male fertility independent of lipidation and autophagy, which would be exciting to study in mammals, but it is challenging due to the presence of 7 paralogs (Jipa, 2021).

    Autophagy has been suggested to contribute to male fertility in mammals via, for example, ensuring proper lipid homeostasis for testosterone production in Leydig cells, but this is clearly not the case in Drosophila. Lipidation-independent functions of Atg8 family proteins have also been reported in mammals. Unlipidated MAP1LC3 proteins are associated with intracellular Chlamydia and it is important for the propagation of bacteria, while inhibition of autophagy enhanced chlamydial growth. Unlipidated MAP1LC3 proteins coat EDEMosomes: ER-derived vesicles transporting ER chaperones including EDEM1 to endosomes for breakdown. Of note, coronaviruses are known to hijack this pathway to generate double-membrane vesicles (DMVs) that aid virus replication. Thus, understanding lipidation-independent functions of Atg8 family proteins has clear medical relevance, especially considering the coronavirus pandemic started at the end of 2019 (Jipa, 2021).

    Taken together, this study showed that Atg8a was important for developmentally programmed removal of larval gastric ceca, for proper pupal development and for the eclosion of adult flies. These were all independent of its lipidation based on analysis of the unlipidatable mutant, likely reflecting an autophagy-independent role of Atg8a in these processes. This study also showed that high expression of Atg8b was required for male fertility independent of its lipidation and autophagy. The new mutant and transgenic animals generated in this study will be useful to further study these exciting phenomena (Jipa, 2021).

    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).

    Snazarus and its human ortholog SNX25 modulate autophagic flux

    Macroautophagy, the degradation and recycling of cytosolic components in the lysosome, is an important cellular mechanism. It is a membrane-mediated process that is linked to vesicular trafficking events. The sorting nexin (SNX) protein family controls the sorting of a large array of cargoes, and various SNXs impact autophagy. To improve understanding of their functions in vivo, all Drosophila SNXs were screened using inducible RNA interference in the fat body. Significantly, depletion of Snazarus (Snz) led to decreased autophagic flux. Interestingly, altered distribution of Vamp7-positive vesicles was observed with Snz depletion, and the roles of Snz were conserved in human cells. SNX25, the closest human ortholog to Snz, regulates both VAMP8 endocytosis and lipid metabolism. Through knockout-rescue experiments, it was demonstrated that these activities are dependent on specific SNX25 domains and that the autophagic defects seen upon SNX25 loss can be rescued by ethanolamine addition. The presence of differentially spliced forms of SNX14 and SNX25 was detected in cancer cells. This work identifies a conserved role for Snz/SNX25 as a regulator of autophagic flux and reveals differential isoform expression between paralogs (Lauzier, 2022).

    Macroautophagy, hereafter termed autophagy, is a crucial homeostatic and stress-responsive catabolic mechanism. Autophagy is characterized by the formation of double-membrane structures, called phagophores, which expand and incorporate cytoplasmic proteins or organelles. These structures ultimately close to form autophagosomes. When mature, the autophagosomes fuse with lysosomes, and autophagosomal content is degraded by lysosomal enzymes and recycled. Hence, autophagy requires an intricate balance between various cellular processes to ensure appropriate cargo selection, and autophagosome formation, maturation and fusion (Lauzier, 2022).

    Although the core signaling pathways controlling autophagy induction in response to stress were rapidly described and are now well understood, the molecular mechanisms controlling autophagosome sealing, maturation and fusion were only defined more recently. Findings in yeast and metazoans have shed light on the molecular machinery required for autophagosome-lysosome fusion and its regulation. Although different proteins are involved in autophagosome-vacuole fusion in yeast and autophagosome-lysosome fusion in metazoans, the overarching principle is conserved and requires the presence of specific soluble N-ethylmaleimide-sensitive factor attachment receptors (SNAREs). In metazoans, syntaxin (STX) is recruited to mature autophagosomes by two hairpin regions, where it forms a Qabc complex with synaptosome associated protein 29 (SNAP29). The STX17-SNAP29 complex then forms a fusion-competent complex with lysosome-localized vesicle associated membrane protein (VAMP). More recently, the Qa SNARE YKT6 v-SNARE homolog (YKT6) was also found to mediate autophagosome-lysosome fusion. YKT6 is recruited to mature autophagosomes and associates with SNAP29. The YKT6-SNAP29 complex interacts with the lysosomal R-SNARE STX7 to mediate fusion . These fusion complexes are conserved, and flies also use these proteins for autophagosome-lysosome fusion. However, unlike in human cells, where STX17 and YKT6 act redundantly in parallel pathways, Ykt6 is epistatic to Syx17 and Vamp7 in flies. SNARE functions are supported by other intracellular factors, which ensure their specificity and rapid action. The small Rab GTPases Ras-related protein RAB7 and RAB2 are important determinants of fusion, as lysosome-localized RAB7 and autophagosome-localized RAB2 interact with the tethering homotypic fusion and vacuole protein sorting (HOPS) complex to bring autophagosomes and lysosomes in close proximity and enable SNARE-mediated fusion. Interestingly, a direct interaction has been observed between STX17 and the HOPS complex, favoring autophagosome-lysosome tethering. The lipid composition of autophagosomes and lysosomes is also an important determinant of fusion. Specific phosphoinositides [PtdIns(3)P, PtdIns(3,5)P2, PtdIns(4)P, and PtdIns(4,5)P2] impact fusion through different mechanisms. Low cholesterol levels affect autophagosome tethering to late endosomes/lysosomes, while increased saturated fatty acid levels or a high-fat diet in mice decrease fusion events. Recently, the phosphatidylserine:phosphatidylethanolamine ratio was also demonstrated to affect autophagosome-lysosome fusion (Lauzier, 2022).

    It is clear that multiple inputs are integrated to regulate the final step of the autophagic process. Accordingly, trafficking events must properly regulate the trafficking of essential SNAREs involved in autophagosome-lysosome fusion, like VAMP8 and STX7, that also mediate various other membrane fusion events. This is also true for the dynamic regulation of the lipid composition of these organelles, given that inappropriate ratios of specific lipids affect autophagic flux. Hence, defining trafficking regulators coordinating the localization of SNAREs, as well as the lipid composition of autophagosomes and lysosomes, is of paramount importance for better understanding of the dynamic link between trafficking and autophagy (Lauzier, 2022).

    One class of endosomal sorting regulators is the sorting nexin (SNX) family. These proteins have phox homology (PX) domains that interact with diverse phosphoinositide species. Many SNXs localize to early endosomes, where they are involved in sorting events. Importantly, a few SNXs play roles in autophagy. SNX18 and SNX4-SNX7 heterodimers control autophagy-related ATG9 trafficking to modulate autophagosome expansion, and SNX5 and SNX6 also indirectly regulate autophagy by modulating cation-independent mannose-6-phosphate receptor sorting, affecting lysosomal functions. In yeast, SNX4 regulates autophagosome-lysosome fusion by controlling endosomal phosphatidylserine levels. These reports highlight the multifaceted roles of SNXs in regulating autophagy. However, SNX involvement in SNARE protein trafficking has not been reported (Lauzier, 2022).

    Using Drosophila as a simple system to screen genes involved in autophagy, this study has identified the sorting nexin Snazarus (Snz) and its human ortholog SNX25 as regulators of the localization and lipid metabolism of Vamp7 and VAMP8, respectively. Using RNA interference (RNAi) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-generated mutants, as well as ethanolamine supplementation, this study showed that loss of Snz decreases autophagic flux. Importantly, it was shown that this effect is independent of the endoplasmic reticulum (ER) localization of SNX25 and that it affects two independent processes - Vamp7/VAMP8 internalization and lipid homeostasis. Altogether, these findings identify Snz and SNX25 as regulators of autophagic flux (Lauzier, 2022).

    This study has uncovered a conserved autophagic function for snz and its ortholog SNX25. Using both RNAi-mediated depletion and CRISPR/Cas9-generated KOs, it was shown that Snz and SNX25 are required for full autophagic flux. The impact on autophagy is unlikely to occur via lysosomal dysfunction, but potentially through a combination of inappropriate Vamp7 (in flies) and VAMP8 (in humans) internalization or trafficking and defective lipid metabolism. Interestingly, the SNX25 PX domain was necessary for VAMP8 uptake, while ER anchoring was dispensable. Furthermore, LC3 accumulation observed upon SNX25 loss could be rescued by SNX25 lacking either its PX/Nexin or ER anchoring domains, and by ETA supplementation. Altogether, the findings uncover the multifaceted effects of SNX25 loss on endocytosis and lipid metabolism, which ultimately affect autophagic flux (Lauzier, 2022).

    To further refine the endosomal sorting regulators involved in autophagy, a targeted RNAi screen was performed of SNXs in the fly fat body and monitored autolysosome formation. Unexpectedly, most SNXs tested caused defects in autolysosome acidification. It is believed that this is a consequence of the wide range of cargos sorted or endocytosed by SNXs. The misrouting of specific cargos could directly or indirectly affect lysosomal function and therefore autolysosome acidification or formation. The results also reveal the potential for complementation between SNXs paralogs in mammalian cells, which may explain why autophagy defects were not observed for most SNXs in genome-wide screens (Lauzier, 2022).

    SNX14 has three paralogs in mammals - SNX13, SNX19 and SNX25. In neural precursor cells derived from patients with SCAR20, SNX14 loss was associated with autophagosome clearance defects. Conversely, weak effects were observed in dermal fibroblasts from patients. As Drosophila have only a single ortholog of these proteins,it was possible to show through multiple approaches that loss of Snz affected autophagosome clearance and led to autophagosome and autophagic cargo [ref(2)P] accumulation. Data in HeLa cells also indicate defective autophagic flux in SNX14- and SNX25-KO cells. The differences between the current results and findings in patient fibroblasts might be due to differential regulation of either paralog expression or mRNA splicing between cell types. It is worth mentioning that, in HeLa cells, increased SNX14 expression was detected upon SNX25 KO (Fig. 2F). Furthermore, given the complementation of SNX25 KO by SNX14 expression, it is conceivable that SNX25 expression could be differentially modulated in various cell types and be able to rescue SNX14-linked autophagic defects (Lauzier, 2022).

    The data indicate defects in the trafficking of Vamp7 and VAMP8 after depletion of Snz and SNX25, respectively. Since the YKT6-SNAP29-STX7 complex can also promote autophagosome-lysosome fusion, it is likely that this complex partially complements the loss of Snz and SNX25, which would explain why their loss did not completely abrogate autophagic flux. Along these lines, differential expression of SNARE complexes between cell types could also account for the variations in penetrance observed between SNX14 studies (Lauzier, 2022).

    How exactly Snz/SNX25 regulates Vamp7/VAMP8 endocytosis or trafficking remains to be defined. It was not possible to directly test Vamp7 trafficking in flies; however, ectopic accumulation of GFP:Vamp7 puncta was observed near or at the PM, suggesting a potential uptake defect. To test this more directly, VAMP8 uptake was assessed in SNX25 KO cells. Interestingly, these cells showed decreased VAMP8 internalization that was dependent on the SNX25 PX domain, which interacts with diphosphorylated phosphoinositides like PtdIns(4,5)P2, which is highly abundant at the PM. Defects were not observed in clathrin-dependent or -independent endocytosis, nor were variations in clathrin recruitment at the PM. Hence, it is unlikely that SNX25 depletion results in VAMP8 trafficking defects by affecting PtdIns(4,5)P2 or PtdIns(3,4)P2 dynamics at the PM. Recently, Snz was demonstrated to bridge PM-ER contact sites to modulate LD formation. Therefore, SNX25 may fulfill a similar function in mammals, bridging PM-ER contact sites to favor VAMP8 internalization. A precedent for the involvement of ER-PM contact sites in endocytosis exists; however, it was possible to rescue VAMP8 internalization in SNX25 KO cells with a transgene lacking its ER-anchoring domains, implying that ER-PM proximity is not required for efficient VAMP8 uptake. This notion is consistent with the known requirement of PICALM for VAMP8 uptake. Surprisingly, no defects were detected in PICALM localization in SNX25 or SNX14/SNX25 KO cells, although close proximity between it and overexpressed SNX25 was observed. VAMP8 can also be internalized through a clathrin-independent pathway stimulated by Shiga toxin. This pathway is dependent on lipid organization and might be perturbed in SNX25 KO cells. An earlier study identified SNX25 as a regulator of transforming growth factor β receptor (TGFβR) endocytosis. However, this study erroneously characterized the ΔTM isoform of SNX25 and showed that overexpression of this short isoform increased TGFβR internalization, while SNX25 knockdown decreased uptake. Thus, Snz/SNX25 might affect the endocytosis of multiple cargos, in addition to Vamp7 and VAMP8 (Lauzier, 2022).

    It is also worth mentioning that the yeast ortholog of snz and SNX25, MDM1, was originally identified as a regulator of endocytic trafficking, thus other aspects of trafficking could be impaired in Snz/SNX25 mutants and be sensitive to protein expression levels. Although the data illustrate decreased internalization of VAMP8 in SNX25 KO cells, the possibility remains that VAMP8, in addition to its uptake defect, could be misrouted on route to autolysosomes. Decreased colocalization was observed between VAMP8 and CD63 in SNX25 KO cells; therefore, defective trafficking cannot be ruled out. Moreover, co-expression of both SNX25 and VAMP8 led to the re-localization of both proteins to large internal vesicles. This effect required the TM region of SNX25, thus it is conceivable that although the short isoform is sufficient for VAMP8 internalization, the longer ER-associated isoform could regulate the endosomal sorting of VAMP8, through potential inter-organellar contact sites or by modulating lipid metabolism (Lauzier, 2022).

    Recent studies have demonstrated important roles for SNX14 in lipid metabolism. SNX14 loss results in saturated fatty acid accumulation and increased sensitivity to lipotoxic stress. Moreover, SNX14, Snz and Mdm1, the yeast ortholog, all regulate LD formation. The functional domains required for SNX14 regulation of LD formation differ from the ones required in SNX25 for VAMP8 uptake; the TM and C-terminal nexin domains of SNX14 are essential for LD localization and regulation, while the PX domain of SNX25 is required for VAMP8 uptake, and its TM domains are dispensable. Interestingly, LD biogenesis, fatty acid trafficking and autophagy are known to intersect. In this context, it is tempting to speculate that Snz and its human orthologs SNX14 and SNX25 could bridge lipid stress and autophagy regulation. Further supporting this hypothesis is the finding that SNX25 loss can be rescued by SNX14 or by either SNX25ΔTM and SNX25ΔPX/Nexin. Moreover, ETA addition, which is predicted to result in higher intracellular phosphatidylethanolamine levels, rescued SNX25 deletion. These rescue experiments highlight that SNX25 loss causes independent phenotypes that culminate in decreased autophagic flux. The effects are likely more potent in flies, since they have a single ortholog and the data show that SNX14 can efficiently rescue SNX25 loss. Concerning the role of SNX25 in lipid metabolism, it is tempting to speculate that it is most probably linked to an effect on lipid saturation and LD biogenesis for four main reasons. First, the C-Nexin region of SNX14 was shown to mediate LD localization, and SNX25 loss could be rescued using a SNX25 mutant deleted of this region, arguing that LD recruitment of SNX25 is dispensable. Second, KO/rescue experiments in HeLa cells were performed in normal growth conditions, where LD biogenesis is minimal, and thus unlikely to affect autophagy. Third, recent findings in U2OS cells identified the PXA region of SNX14 as important in regulating lipid saturation and ER stress in response to saturated lipid accumulation. As the PXA was conserved in the two rescue constructs used for autophagy rescue, it is plausible that SNX25 somehow affects lipid homeostasis and thus autophagosome-lysosome fusion. Moreover, recent findings illustrated the importance of the PE ratio in membrane fusion, and SNX14 deletion leads to increased phosphatidylserine levels as in SNX4 yeast mutants (Ma et al., 2018). This intriguing possibility warrants further studies to identify the specific determinants that mediate the action of SNX25 in endocytosis versus lipid homeostasis (Lauzier, 2022).

    Another possibility to consider is that SNX25 may encode ba lipid clustering or transport domain that could help concentrate lipids or move them between organelles in a manner that support functional autophagy. In support of this, recent work using Alphafold2 structural predictions suggest that the Nexin-C and PXA domains of the yeast SNX25 ortholog Mdm1 fold together to create a large spherical domain with a hydrophobic channel that could, in principle, ferry lipids between organelles at organelle contacts. Such a domain could enable SNX25 to localize to various intracellular sites, and cluster and/or transport lipids to support functional autophagy. SNX14 is predicted to contain this domain arrangement as well and this might explain why it can rescue SNX25 loss. In this model, loss of SNX25 would alter lipid homeostasis and subcellular distribution, leading to defects in Vamp7/VAMP8 trafficking and functional autophagy. The molecular details for this process, however, remain to be addressed (Lauzier, 2022).

    The observation that various isoforms of SNX14 and SNX25 are expressed in cells is intriguing. This raises the possibility of functional pools of SNX14 and SNX25, with the longer ER-anchored isoform regulating LD biogenesis and the shorter isoforms regulating other processes, like trafficking and autophagy. It is worth noting, however, that although this study provides evidence from ddPCR experiments, it was not possible to demonstrate differential splicing at the protein level because of a lack of isoform-specific antibodies. Isoform expression may be controlled by modulating splicing in response to stress, as has been observed for multiple genes. Alternatively, different transcription factors may favor the expression of certain isoforms. RNA-sequencing datasets from Drosophila do not contain different Snz isoforms, suggesting that a single isoform regulates both LD biogenesis and autophagy (Lauzier, 2022).

    In summary, this study has identified a new role for snz and its ortholog SNX25 in autophagy regulation through effects on Vamp7/VAMP8 internalization and lipid metabolism. Moreover, differentially expressed isoforms of SNX14 and SNX25 were described in cancer cells. Based on thesd results and those of previous studies,it is propose that Snz and SNX25 finetune the endocytosis/trafficking of Vamp7 and VAMP8 and potentially regulate the lipid composition of endolysosomes to coordinate the autophagy level with the demands of the cell. It will be interesting to define how these functions differ between various genes and isoforms, and how they are affected by different stressors (Lauzier, 2022).

    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).

    Loss of Hsp67Bc leads to autolysosome enlargement in the Drosophila brain

    Hsp67Bc is a small heat shock protein found in Drosophila melanogaster. Apart from performing a function (common for all small heat shock proteins) of preventing aggregation of misfolded proteins, it is involved in macroautophagy regulation alongside the Starvin protein. Overexpression of the D. melanogaster Hsp67Bc gene has been shown to stimulate macroautophagy in S2 cell culture. Nonetheless, it has been unknown how the absence of the Hsp67Bc gene may affect it. The effect of Hsp67Bc gene deletion was studied on the macroautophagy induced by the pathogenic Wolbachia wMelPop strain in D. melanogaster. Wolbachia was detected inside autophagic vacuoles in fly neurons, thereby proving that these endosymbionts were being eliminated via macroautophagy. Nevertheless, no difference was registered in brain bacterial load between Hsp67Bc-null and control flies at all tested stages of ontogenesis. Moreover, the abundance of autophagic vacuoles was similar between neurons of the mutant and control flies, yet the cross-sectional area of autolysosomes on ultrathin sections was more than 1.5-fold larger in Hsp67Bc-null fly brains than in the control line. These findings suggest that the product of the Hsp67Bc gene does not participate in the initiation of endosymbiont-induced macroautophagy but may mediate autophagosome maturation: the deletion of the Hsp67Bc gene leads to the increase in autolysosome size (Malkeyeva, 2021).

    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).

    Identifying and monitoring neurons that undergo metamorphosis-regulated cell death (metamorphoptosis) by a neuron-specific caspase sensor (Casor) in Drosophila melanogaster

    Activation of caspases is an essential step toward initiating apoptotic cell death. During metamorphosis of Drosophila melanogaster, many larval neurons are programmed for elimination to establish an adult central nervous system (CNS) as well as peripheral nervous system (PNS). However, their neuronal functions have remained mostly unknown due to the lack of proper tools to identify them. To obtain detailed information about the neurochemical phenotypes of the doomed larval neurons and their timing of death, a new GFP-based caspase sensor (Casor) was generated that is designed to change its subcellular position from the cell membrane to the nucleus following proteolytic cleavage by active caspases. Ectopic expression of Casor in vCrz and bursicon, two different peptidergic neuronal groups that had been well-characterized for their metamorphic programmed cell death, showed clear nuclear translocation of Casor in a caspase-dependent manner before their death. Similar events in some cholinergic neurons from both CNS and PNS. Moreover, Casor also reported significant caspase activities in the ventral and dorsal common excitatory larval motoneurons shortly after puparium formation. These motoneurons were previously unknown for their apoptotic fate. Unlike the events seen in the neurons, expression of Casor in non-neuronal cell types, such as glial cells and S2 cells, resulted in the formation of cytoplasmic aggregates, preventing its use as a caspase sensor in these cell types. Nonetheless, these results support Casor as a valuable molecular tool not only for identifying novel groups of neurons that become caspase-active during metamorphosis but also for monitoring developmental timing and cytological changes within the dying neurons (Lee, 2018).

    Combinatorial action of Grainyhead, Extradenticle and Notch in regulating Hox mediated apoptosis in Drosophila larval CNS

    Hox mediated neuroblast apoptosis is a prevalent way to pattern larval central nervous system (CNS) by different Hox genes, but the mechanism of this apoptosis is not understood. Studies with Abdominal-A (Abd-A) mediated larval neuroblast (pNB) apoptosis suggests that AbdA, its cofactor Extradenticle (Exd), a helix-loop-helix transcription factor Grainyhead (Grh), and Notch signaling transcriptionally contribute to expression of RHG family of apoptotic genes. Grh, AbdA, and Exd were found to function together at multiple motifs on the apoptotic enhancer. In vivo mutagenesis of these motifs suggest that they are important for the maintenance of the activity of the enhancer rather than its initiation. Exd function is independent of its known partner homothorax in this apoptosis. Some findings were extended to Deformed expressing region of sub-esophageal ganglia where pNBs undergo a similar Hox dependent apoptosis. A mechanism is proposed where common players like Exd-Grh-Notch work with different Hox genes through region specific enhancers to pattern respective segments of larval central nervous system (Khandelwal, 2017).

    Trans-generational transmission of altered phenotype resulting from flubendiamide-induced changes in apoptosis in larval imaginal discs of Drosophila melanogaster

    The eye and wing morphology of Drosophila maintain unique, stable pattern of genesis from eye and wing imaginal discs. Increased apoptosis in discs was found to be associated with flubendiamide (fluoride containing insecticide) exposure in Drosophila larvae. The chemical fed larvae on attaining adulthood revealed alterations in morphology and symmetry of their compound eyes and wings through scanning electron microscopy. Nearly 40% and 30% of flies (P generation) demonstrated alterations in eyes and wings respectively. Transmission electron microscopic study also established variation in the rhabdomere and pigment cell orientation as well as in the shape of the ommatidium. Subsequent SEM study with F1 and F2 generation flies also revealed structural variation in eye and wing. Decrease in percentage of altered eye and wing phenotype was noted in subsequent generations. Thus, flubendiamide was found to increase apoptosis in larvae and thereby cause morphological alteration in the adult Drosophila. This study further demonstrated trans-generational transmission of altered phenotype in three subsequent generations of Drosophila (Sarkar, 2017).

    Overexpression of histone methyltransferase NSD in Drosophila induces apoptotic cell death via the Jun-N-terminal kinase pathway

    The nuclear receptor-binding SET domain protein gene (NSD) family encodes a group of highly conserved SET domain-containing histone lysine methyltransferases that are important in multiple aspects of development in various organisms. The association of NSD1 duplications has been reported with growth retardation diseases in humans. To gain insight into the molecular mechanisms by which the overexpression of NSD1 influences the disease progression, this study examined the gain-of-function mutant phenotypes of the Drosophila NSD using the GAL4/UAS system. Ubiquitous overexpression of NSD in the fly caused developmental delay and reduced body size at the larval stage, resulting in pupal lethality. Moreover, targeted overexpression in various developing tissues led to significant phenotype alterations, and the gain-of-function phenotypes were rescued by NSD RNAi knockdown. NSD overexpression not only enhanced the transcription of pro-apoptotic genes but also activated caspase. The atrophied phenotype of NSD-overexpressing wing was strongly suppressed by a loss-of-function mutation in hemipterous, which encodes a Drosophila Jun N-terminal kinase. Taken together, these findings suggest that NSD induces apoptosis via the activation of JNK, and thus contributes to the understanding of the molecular mechanisms involved in NSD1-related diseases in humans (Jeong, 2018).

    Plasma membrane localization of apoptotic caspases for non-apoptotic functions

    Caspases are best characterized for their function in apoptosis. However, they also have non-apoptotic functions such as apoptosis-induced proliferation (AiP), where caspases release mitogens for compensatory proliferation independently of their apoptotic role. This study reports that the unconventional myosin, Myo1D, which is known for its involvement in left/right development, is an important mediator of AiP in Drosophila. Mechanistically, Myo1D translocates the initiator caspase Dronc to the basal side of the plasma membrane of epithelial cells where Dronc promotes the activation of the NADPH-oxidase Duox for reactive oxygen species generation and AiP in a non-apoptotic manner. It is proposed that the basal side of the plasma membrane constitutes a non-apoptotic compartment for caspases. Finally, Myo1D promotes tumor growth and invasiveness of the neoplastic scrib Ras(V12) model. Together, these studies have identified a new function of Myo1D for AiP and tumorigenesis and reveal a mechanism by which cells sequester apoptotic caspases in a non-apoptotic compartment at the plasma membrane (Amcheslavsky, 2018).

    Under stress conditions, when a large number of cells are dying, there is a need for compensatory proliferation to replace the lost cells with new cells. Work using several model organisms has shown that, under these conditions, apoptotic cells can release mitogenic signals that induce proliferation of surviving cells for the replacement of dying cells. Because apoptotic cells are actively triggering this type of compensatory proliferation, this process has been termed apoptosis-induced proliferation (AiP) (Amcheslavsky, 2018).

    Caspases are Cys proteases that are the main effectors of apoptosis. They are produced as inactive zymogens with a prodomain and after processing a large and small subunit. There are initiator and effector caspases. Initiator caspases carry protein/protein interacting motifs in their prodomains, which mediate their incorporation into large multimeric protein complexes. For example, the mammalian initiator caspase-9 is recruited into the Apaf-1 apoptosome, while its Drosophila ortholog Dronc forms the apoptosome with the Apaf-1 homolog Dark. Effector caspases such as mammalian caspase-3, or Drosophila DrICE and Dcp-1, are proteolytically processed by activated initiator caspases and mediate the apoptotic process (Amcheslavsky, 2018).

    In addition to apoptosis, caspases are also mediating AiP. They trigger the release of Wnt, bone morphogenetic protein (BMP)/transforming growth factor β (TGF-β), epidermal growth factor (EGF), and Hedgehog mitogens for AiP. This has been best studied for the Drosophila initiator caspase Dronc using the 'undead' AiP model in which apoptotic signaling is induced by expression of upstream cell death factors such as hid, but the execution of apoptosis is blocked by co-expression of the effector caspase inhibitor p35, thus rendering cells in an undead condition. Because P35 inhibits apoptosis, but not Dronc, Dronc can still mediate non-apoptotic functions such as AiP. When hid and p35 are co-expressed using the ey-Gal4 driver (ey > hid,p35), which is expressed in epithelial cells of eye imaginal discs, Dronc continuously signals for AiP and triggers hyper-proliferation. Consequently, the discs are enlarged and the resulting heads of the adult flies are overgrown . In genetic screens, screening was carried out for suppressors of the overgrowth phenotype of undead (ey > hid,p35) adult heads to identify genes and mechanisms involved in AiP (Amcheslavsky, 2018).

    Mechanistically, this study showed that, in undead cells, Dronc stimulates the NADPH-oxidase Duox for the production of extracellular reactive oxygen species (eROS). eROS recruit and activate hemocytes, Drosophila immune cells similar to macrophages, to the undead imaginal disc. In turn, hemocytes release the tumor necrosis factor-like ligand Eiger, which induces JNK activity in epithelial disc cells. JNK promotes the expression of the apoptotic genes reaper and hid, which initiate a positive feedback loop to maintain undead signaling (Fogarty, 2016). In addition, it induces the release of the mitogens Wingless (Wg), a Wnt-like gene in Drosophila, decapentaplegic, a BMP/TGF-β homolog, and Spitz, an EGF ligand, which all promote AiP (Amcheslavsky, 2018).

    In addition to undead AiP, there is also 'genuine' AiP, during which dying cells complete the apoptotic process, and the response of the affected tissue to replace the dying cells is examined. In contrast to undead AiP, genuine AiP does not promote overgrowth. Therefore, although most genes identified in undead AiP also have important roles in genuine AiP, there must be differences between the two AiP models. In any case, genuine AiP is used as a model of tissue regeneration, while the hyper-proliferation of undead AiP serves as a tumorigenic model (Amcheslavsky, 2018).

    Class I unconventional myosins are conserved actin-based motor proteins, composed of the N-terminal head (motor) region with an ATP binding motif (including P-, switch1-, and switch2 loops) and an actin-binding domain, a neck region characterized by two to three IQ motifs, and a C-terminal tail domain that interacts with phospholipids at membranes. Mammals have eight class I myosins, Drosophila has three, Myosin 1D (Myo1D, also known as Myo31DF), MyoIC (Myo61F), and Myo95E. While Myo1D and Myo1C are involved in left/right (L/R) development of visceral organs, the function of Myo95E is unknown (Amcheslavsky, 2018).

    Although Drosophila is a bilateral organism, certain visceral organs such as the gut and the coiling of the spermiducts around the gut, which occurs in a morphogenetic movement termed male terminalia rotation, display L/R asymmetry. In Myo1D mutants, the chirality of these asymmetric organs and movements are reversed. For example, the male terminalia rotation during pupal development, which, in wild-type, occurs for 360° in clockwise (dextral) orientation, proceeds in Myo1D mutants sinistrally, defining Myo1D as dextral determinant. Myo1D engages the actin cytoskeleton and adherens junctions for this movement (Amcheslavsky, 2018).

    Overexpression of Myo1C antagonizes the dextral activity of Myo1D by displacing it from adherens junctions. However, the loss-of-function phenotype of Myo1C did not confirm this antagonizing function. Instead, while Myo1C single mutants do not display any L/R defect, the Myo1C Myo1D double mutant has a stronger sinistral male terminalia phenotype than Myo1D mutants indicating that Myo1C has a partially redundant dextral activity with Myo1D (Amcheslavsky, 2018).

    It has long been known that genes in the apoptosis pathway, such as hid, dronc, and drICE, are also involved in male terminalia rotation in Drosophila. Indeed, localized apoptotic activity is required for this L/R process. How Myo1D and the apoptosis pathway interact for male terminalia rotation is not very well understood. Interestingly, mutants of the JNK signaling pathway or overexpression of puckered, an inhibitor of JNK activity, also display defects in male terminalia rotation (Amcheslavsky, 2018).

    This study reports that Myo1D is an essential component of AiP in the undead model. Genetic inactivation of Myo1D strongly suppresses ey > hid,p35-induced overgrowth of the head capsule, while overexpression of Myo1D enhances it. Myo1D promotes the generation of ROS by Duox for AiP signaling. Further mechanistic analysis reveals that Myo1D is required for membrane localization of Dronc, specifically to the basal side of the plasma membrane of undead epithelial disc and salivary gland cells. Here, Dronc exerts a non-apoptotic function resulting in Duox activation. It is proposed that the basal side of the plasma membrane constitutes a non-apoptotic compartment that allows non-apoptotic processes of Dronc and potentially other caspases to occur. Therefore, in addition to the dextral activity of Myo1D, this study identified a second function of Myo1D for the control of apoptosis-induced proliferation (Amcheslavsky, 2018).

    Mechanistically, it was found that Myo1D is involved in the localization of the initiator caspase Dronc to the basal side of the plasma membrane of undead DP disc and SG cells. Myo1D interacts with Dronc, suggesting that it may directly translocate Dronc to the plasma membrane. However, Myo1D does not appear to be a cleavage target of the caspase Dronc (Amcheslavsky, 2018).

    The observed localization of Dronc to the basal side of the plasma membrane in undead DP cells is critical for the mechanism of AiP. Undead cells attract hemocytes to the discs in a Dronc- and Duox-dependent manner. However, that occurs at the basal side of DP cells of imaginal discs because the basal side is exposed to the hemolymph that contains circulating hemocytes, while the apical side faces the lumen between the DP and the PM. Consistently, there is also an enrichment of Duox at the basal side of the plasma membrane. Therefore, in order to be able to activate Duox for ROS generation and hemocyte activation, Dronc needs to be specifically present at the basal side of the plasma membrane (Amcheslavsky, 2018).

    It has long been known that caspases, including Dronc, have non-apoptotic functions in addition to their well characterized role in apoptosis. This paper reveals one mechanism by which cells may activate a caspase (Dronc) without the detrimental consequences of apoptosis. The sequestration of Dronc to the basal side of the plasma membrane in a Myo1D-dependent manner and the low abundance of Dronc's apoptotic partner Dark at the plasma membrane may ensure localized and controlled apoptosome activity which is sufficient for AiP, but not for killing cells. Alternatively, apoptotic substrates needed for the execution of apoptosis may not be present at the plasma membrane or in insufficient amount to pass the apoptotic threshold (Amcheslavsky, 2018).

    While this study addressed the role of membrane localization of Dronc under undead conditions, recently membrane-localized Dronc was shown in SGs under normal conditions, which explains the membrane localization of Dronc at control SGs. Here, membrane-localized Dronc is required for F-actin cytoskeleton dismantling at the end of larval development in a non-apoptotic manner. In addition to the plasma membrane, the outer mitochondrial membrane has been shown to provide a non-apoptotic platform for caspase activation, in this case during sperm maturation. Therefore, membranes in general may provide a local environment for non-apoptotic caspase activities (Amcheslavsky, 2018).

    The membrane localization of Dronc in SGs is mediated by Tango7, which has previously been implicated in spermatid maturation. As mentioned above, membrane-localized Dronc is required for dismantling of the cortical F-actin cytoskeleton in SGs of late larvae. However, while Tango7 RNAi blocks actin dismantling, Myo1D RNAi does not , suggesting that the roles of Tango7 and Myo1D for membrane localization of Dronc are different from each other. That also explains why in undead SGs the membrane localization of Dronc strongly increases in a Myo1D-dependent manner. Unfortunately, it was not possible to test if Tango7 is involved in AiP. Tango7 RNAi in eye imaginal discs results in complete loss of the disc. Tango7 encodes the homolog of eukaryotic translation initiation factor 3m (eIF3m), suggesting that it may also have an important requirement for protein translation, explaining the loss of the eye disc by Tango7 RNAi (Amcheslavsky, 2018).

    In addition to Myo1D and Tango7, there is at least one other factor, Crinkled (Ck), which directs Dronc to non-apoptotic functions. Ck bridges the interaction between Dronc and the kinase Shaggy/glycogen synthase kinase beta (GSK-β), resulting in the selective activation of Shaggy/GSK-β, which then promotes non-apoptotic activities such as the specification of scutellar bristles, border cell migration, and correct branching of the aristae. Interestingly, Ck encodes another unconventional myosin, a member of the class VII myosin family, potentially suggesting that other myosins may also direct non-apoptotic functions to caspases (Amcheslavsky, 2018).

    Myo1D and the apoptotic machinery have been linked to male terminalia rotation, an L/R process during pupal development. Indeed, apoptosis is required for Myo1D-dependent male terminalia rotation. It is unknown how Myo1D interacts with the apoptotic machinery to direct this L/R movement. In future studies, it will be interesting to examine if the Myo1D-dependent mechanism identified here for AiP also applies to male terminalia rotation or whether a separate mechanism exists in this context (Amcheslavsky, 2018).

    Myo1D not only localizes Dronc to the plasma membrane, it also stabilizes it. Dronc is activated in undead cells, and activated Dronc is subject of increased protein degradation. Thus, Myo1D prevents degradation of Dronc by changing its subcellular localization to the plasma membrane (Amcheslavsky, 2018).

    Myo1D has a very strong requirement for AiP in the undead model, and a requirement in the scrib-/-RasV12 tumorigenesis model, yet it does not appear to play any significant role in genuine AiP. In fact, Myo1D is the first gene identified that is essential for the hyper-proliferation of undead AiP, but not required for the regeneration of genuine AiP. The mechanism revealed in this paper provides an explanation for this behavior. During genuine AiP, cells are allowed to undergo apoptosis, which requires cytosolic Dronc activity. Although ROS are generated during genuine AiP, the origin of these ROS has not been determined and may not require the plasma membrane-localized Duox. Therefore, a key difference between genuine AiP and undead AiP, and potentially between other regenerative versus tumorigenic models, may be the altered localization of Dronc to a non-apoptotic compartment at the plasma membrane, and a shift from balanced apoptosis and proliferation to dominant proliferation. The next big question will be to examine what exactly is prompting Myo1D to drive this re-localization of Dronc under sustained undead conditions, but not under the limited regenerative conditions of the genuine AiP models, and whether that answer provides any insight into the cancer versus wound healing models (Amcheslavsky, 2018).

    In conclusion, in addition to its role in L/R development, this study identified a second function of Myo1D for AiP and tumorigenesis. The basal side of the plasma membrane was identified as a non-apoptotic environment for caspase function. In future work, it will be important to identify the mechanisms by which Dronc mediates its non-apoptotic functions at the plasma membrane for AiP and other cellular processes that require membrane localization of Dronc and other caspases (Amcheslavsky, 2018).

    A comprehensive in vivo screen for anti-apoptotic miRNAs indicates broad capacities for oncogenic synergy

    microRNAs (miRNAs) are ~21-22 nucleotide (nt) RNAs that mediate broad post-transcriptional regulatory networks. However, genetic analyses have shown that the phenotypic consequences of deleting individual miRNAs are generally far less overt compared to their misexpression. This suggests that miRNA deregulation may have broader phenotypic impacts during disease situations. This concept was explored in the Drosophila eye, by screening for miRNAs whose misexpression could modify the activity of pro-apoptotic factors. Via unbiased and comprehensive in vivo phenotypic assays, this study identified an unexpectedly large set of miRNA hits that can suppress the action of pro-apoptotic genes hid and grim. Secondary assays were used to validate that a subset of these miRNAs can inhibit irradiation-induced cell death. Since cancer cells might seek to evade apoptosis pathways, this situation was modeled by asking whether activation of anti-apoptotic miRNAs could serve as "second hits". Indeed, while clones of the lethal giant larvae (lgl) tumor suppressor are normally eliminated during larval development, this study found that diverse anti-apoptotic miRNAs mediate the survival of lgl mutant clones in third instar larvae. Notably, while certain anti-apoptotic miRNAs can target apoptotic factors, most of the screen hits lack obvious targets in the core apoptosis machinery. These data highlight how a genetic approach can reveal distinct and powerful activities of miRNAs in vivo, including unexpected functional synergies during disease or cancer-relevant settings (Bejarano, 2021).

    Effects of cadmium on oxidative stress and cell apoptosis in Drosophila melanogaster larvae

    With the increase of human activities, cadmium (Cd) pollution has become a global environmental problem affecting biological metabolism in ecosystem. Cd has a very long half-life in humans and is excreted slowly in organs, which poses a serious threat to human health. In order to better understand the toxicity effects of cadmium, third instar larvae of Drosophila melanogaster (Canton-S strain) were exposed to different concentrations (1.125 mg/kg, 2.25 mg/kg, 4.5 mg/kg, and 9 mg/kg) of cadmium. Trypan blue staining showed that intestinal cell damage of Drosophila larvae increased and the comet assay indicated significantly more DNA damage in larvae exposed to high Cd concentrations. The nitroblue tetrazolium (NBT) experiments proved that content of reactive oxygen species (ROS) increased, which indicated Cd exposure could induce oxidative stress. In addition, the expression of mitochondrial adenine nucleotide transferase coding gene (sesB and Ant2) and apoptosis related genes (Debcl, hid, rpr, p53, Sce and Diap1) changed, which may lead to increased apoptosis. These findings confirmed the toxicity effects on oxidative stress and cell apoptosis in Drosophila larvae after early cadmium exposure, providing insights into understanding the effects of heavy metal stress in animal development (Yang, 2022).

    Wg/Wnt1 and Erasp link ER stress to proapoptotic signaling in an autosomal dominant retinitis pigmentosa model

    The endoplasmic reticulum (ER) is a subcellular organelle essential for cellular homeostasis. Perturbation of ER functions due to various conditions can induce apoptosis. Chronic ER stress has been implicated in a wide range of diseases, including autosomal dominant retinitis pigmentosa (ADRP), which is characterized by age-dependent retinal degeneration caused by mutant rhodopsin alleles. However, the signaling pathways that mediate apoptosis in response to ER stress remain poorly understood. In this study, an unbiased in vivo RNAi screen was performed with a Drosophila ADRP model and found that Wg/Wnt1 mediated apoptosis. Subsequent transcriptome analysis revealed that ER stress-associated serine protease (Erasp), which has been predicted to show serine-type endopeptidase activity, was a downstream target of Wg/Wnt1 during ER stress. Furthermore, knocking down Erasp via RNAi suppressed apoptosis induced by mutant rhodopsin-1 (Rh-1(P37H)) toxicity, alleviating retinal degeneration in the Drosophila ADRP model. In contrast, overexpression of Erasp resulted in enhanced caspase activity in Drosophila S2 cells treated with apoptotic inducers and the stabilization of the initiator caspase Dronc (Death regulator Nedd2-like caspase) by stimulating DIAP1 (Drosophila inhibitor of apoptosis protein 1) degradation. These findings helped identify a novel cell death signaling pathway involved in retinal degeneration in an autosomal dominant retinitis pigmentosa model (Park, 2023).

    bfc, a novel Serpent co-factor for the expression of croquemort, regulates efferocytosis in Drosophila melanogaster

    Efferocytosis is the process by which phagocytes recognize, engulf, and digest (or clear) apoptotic cells during development. Impaired efferocytosis is associated with developmental defects and autoimmune diseases. In Drosophila melanogaster, recognition of apoptotic cells requires phagocyte surface receptors, including the scavenger receptor CD36-related protein, Croquemort (Crq, encoded by crq). In fact, Crq expression is upregulated in the presence of apoptotic cells, as well as in response to excessive apoptosis. This study identified a novel gene bfc (booster for croquemort), which plays a role in efferocytosis, specifically the regulation of the crq expression. Bfc protein interacts with the zinc finger domain of the GATA transcription factor Serpent (Srp), to enhance its direct binding to the crq promoter; thus, they function together in regulating crq expression and efferocytosis. Overall, this study shows that Bfc serves as a Srp co-factor to upregulate the transcription of the crq encoded receptor, and consequently boosts macrophage efferocytosis in response to excessive apoptosis. Therefore, this study clarifies how phagocytes integrate apoptotic cell signals to mediate efferocytosis (Zheng, 2021).

    Apoptosis is a developmentally programmed cell death process in multicellular organisms essential for the removal of excessive or harmful cells; whereby apoptotic cells (ACs) are swiftly removed by phagocytes to prevent the release of toxins and induction of inflammation, a process crucial for organ formation, tissue development, homeostasis, and normal immunoregulation. In fact, defects in AC clearance (efferocytosis) can lead to the development of various inflammatory and autoimmune diseases. During efferocytosis, the effective clearance of ACs is accomplished through the recognition and binding of engulfment receptors or bridging molecules on the surface of phagocytes to 'eat me' signals exposed on the surface of ACs. After receptor activation, downstream signals trigger actin cytoskeleton rearrangement and membrane extension around the ACs to form phagosomes. Finally, mature phagosomes fuse with lysosomes to form phagolysosomes, where the internalized ACs are ultimately digested and cleared (Zheng, 2021).

    Since efferocytosis is conserved throughout evolution, it has been studied not only in mammals but also in Drosophila melanogaster. Of note, in D. melanogaster, ACs are removed by non-professional phagocytes, such as epithelial cells and professional phagocytes, such as macrophages and glial cells. Importantly, Drosophila macrophages perform similar functions to those of mammalian macrophages; they participate in both the phagocytosis of ACs and pathogens. Several engulfment receptors have been identified as key players in the recognition and removal of ACs in Drosophila. Franc and colleagues first characterized Croquemort (Crq), a Drosophila CD36-related receptor required by macrophages to engulf ACs. Additionally, Draper (Drpr, a homolog of CED-1/MEGF10) also mediates AC clearance in both glia and macrophages; JNK signaling plays a role in priming macrophages to rapidly respond to injury or microbial infections. Of note, Drpr and its adapter Dmel\Ced-6 (GULP homolog) also seemed important for axon pruning and the engulfment of degenerating neurons by glial cells. The Src tyrosine kinase Src42A (Frk homolog) promotes Drpr phosphorylation and its association with another soluble tyrosine kinase, Shark (ZAP70 homolog), which in turn activates the Drpr pathway. In addition to Drpr, Six-Microns-Under (SIMU) [10] and integrin αPS3 [21] contribute to efferocytosis. SIMU, a Nimrod family cell surface receptor, functions upstream of Drpr to mediate the recognition and clearance of ACs as well as of non-apoptotic cells at wound sites through the recognition of phosphatidylserine (PS). Importantly, the transcriptional factor Serpent (Srp), a GATA factor homolog, was recently found to be required for the efficient phagocytosis of ACs in the context of Drosophila embryonic macrophages and acted via the regulation of SIMU, Drpr, and Crq (Zheng, 2021).

    Searching for other genes required for efferocytosis, this study performed transcriptomic analysis (RNA-seq) and RNAi screening, and discovered 12 genes required for AC clearance in Drosophila S2 cells. In particular, a novel gene, bfc (booster for croquemort), involved in efferocytosis that encodes a specific regulatory factor for the crq transcriptional expression, both in vitro and in vivo. Importantly, this study demonstrated that the GATA factor Srp directly binds to the crq promoter, while Bfc strengthens this binding by interacting with the Srp zinc finger domain. Therefore, a model is proposed in which Bfc cooperates with Srp to enhance crq expression and subsequently induce efferocytosis in D. melanogaster (Zheng, 2021).

    In mammals, ACs are recognized by CD36, one of the several phagocyte cell surface receptors, with the AC surface molecules serving as cognate 'eat-me' signals/ligands. ACs also secrete molecules that attract distant phagocytes and modulate the immune response or phagocytic receptor activity. However, the mechanisms underlying this effect remain unclear. Crq is a CD36-related scavenger receptor in Drosophila and is expressed immediately after the onset of apoptosis in embryonic macrophages. The expression of Crq is regulated by the extent of apoptosis, although the regulatory mechanisms by which ACs control the expression of Crq and subsequently induce phagocytosis in embryonic macrophages have not been described (Zheng, 2021).

    This study has revealed a novel protein, Bfc (Booster for Crq), that plays a key role in efferocytosis via specifically regulating the expression of crq in a manner dependent on the extent of apoptosis. Bfc interacts with the zinc finger domain of the transcription factor Srp as a cofactor to enhance the binding of Srp to the crq promoter, leading to the upregulation of crq expression and the consequent induction of efferocytosis in Drosophila melanogaster. Importantly, the data reveal the molecular mechanisms by which ACs affect Crq expression, as well as how the phagocytic ability of embryonic macrophages is boosted in the presence of excessive apoptosis (Zheng, 2021).

    This study found that in S2 cells, the ACs induced the transcriptional upregulation of crq. In vivo, the macrophages developed as early as the first wave of developmentally programmed apoptosis began at embryogenic stage 11, when the expression of crq was activated and subsequently became widespread throughout the embryo. Importantly, these results are similar to the regulatory mechanisms associated with the expression of other phagocytic receptors, such as Drpr and integrin. For instance, studies showed that AC engulfment rapidly triggers an intracellular calcium burst followed by increased levels of drpr transcripts in Drosophila macrophages; similarly, Draper and integrins become apically enriched soon after the engulfment of apoptotic debris in epithelial follicle cells (Zheng, 2021).

    That the expression of crq was elevated early after the co-culture of ACs and S2 cells, but gradually decreased to the basal levels as efferocytosis continued, suggesting that the regulation of AC clearance and crq expression follow a similar pattern. It was demonstrated that most AC samples added to live S2 cells were composed of apoptotic cells rather than necrotic cells. However, the upregulated expression of genes in response to the presence of a few necrotic cells cannot eliminated. Indeed, based on transcriptome analysis, 12 genes were identified that are required for AC clearance, which was confirmed by subsequent efferocytosis assays using their individual knockdown in S2 cells. Interestingly, among the 12 genes, two were related to innate immunity. CecA1, regulated at the transcriptional level encodes an antibacterial peptide, as well as a secreted protein that mediates the activation of the Toll pathway during bacterial infection. This result may contradict the discreet nature of the apoptotic process. However, ATPs released by bacteria are known to mediate inflammation, and the toll-like receptor 4 (TLR4) is activated by ACs to promote dendritic cell maturation and innate immunity in human monocyte-derived dendritic cells. These results indicate that innate immune pathways are activated in the presence of ACs, and may contribute to their recognition or clearance in Drosophila (Zheng, 2021).

    Among these 12 genes, CG9129 (bfc) and CG30172 regulated the expression of crq and hence, efferocytosis. Further studies must be performed to elucidate the role of CG30172 in efferocytosis. On the other hand, The role played by bfc in efferocytosis as well as the underlying mechanism was clearly dissected. Using several different experimental approaches, this study demonstrated that bfc regulates crq expression in response to excessive apoptosis. First, bfc RNAi treatment decreased the crq expression levels in S2 cells exposed to ACs, but not in the absence of ACs. Second, the increase in crq transcription was proportional to the extent of apoptosis in embryos, which was blocked by the loss of bfc. Notably, other phagocytic receptors have been reported to be activated by dying cells. The integrin heterodimer αPS3/βPS can be enriched in epithelial follicle cells after the engulfment of dying germline cells. In addition, Drpr expression increases in follicle and glial cells, which activates the downstream JNK signaling during the clearance of apoptotic germline cells and neurons, respectively. Collectively, the available scientific literature suggests that the expression of phagocytic receptors can be stimulated by the presence of excessive ACs to improve the phagocytic activity of macrophages or epithelial cells in different tissues (Zheng, 2021).

    Bioinformatics analysis of the conserved domains and gene structure indicated that Bfc does not likely function directly as a transcription factor. This study identified Srp as a Bfc interaction partner using yeast two-hybrid and Co-IP analyses. Shlyakhover (2018) reported that Srp is required for the expression of SIMU, Drpr, and Crq receptors in embryonic macrophages; however, the current results demonstrated that bfc only affects the expression of crq expression through interaction with Srp, with no impact on the expression of several other genes. A plausible hypothesis for this phenotype is that Bfc assistance for Srp binding to the promoters of simu and drpr, may have limited effects. Thus, the results suggest that Bfc may regulate the Crq expression levels in the first wave of AC recognition via binding to Srp, whereas other regulatory factors participate in the Srp-mediated regulation of Drpr and SIMU (Zheng, 2021).

    Srp directly binds to the DNA consensus sequence GATA of the crq promoter via its highly conserved Cys-X2-Cys-X17-Cys-X2-Cys zinc finger binding domain (C4 motif). Meanwhile, Srp also interacts with Bfc through its zinc finger domain; curiously, while the mutation of the C4 motif did not affect the latter interaction, it completely blocked the former. Importantly, it was also shown that mutation in the GATA site abolished the expression of the crq in Drosophila embryo macrophages. As a potential Srp cofactor, Bfc increased the ability of Srp to bind to the crq promoter, while bfc knockdown inhibited the crq transcriptional activity. Ush (homolog of FOG-2 in Drosophila), a cofactor of GATA transcriptional factors, can bind Srp and limit crystal cell production during Drosophila blood cell development. Interestingly, genetic studies have demonstrated that Ush acts with Srp to maintain the pluripotency of hemocyte progenitors and suppresses their differentiation. Ush was reported to repress crq expression by interacting with the isoform of Srp, SrpNC (with two GATA zinc finger) while the other isoform of Srp, SrpC (with one GATA zinc finger) induced crq expression, which may indicate Bfc and Ush act on different isoforms of Srp to regulate crq expression by opposite mechanisms (Zheng, 2021).

    Although the results elucidate several factors that contribute to efferocytosis in Drosophila embryos, some mechanistic details remain unresolved; for instance, how ACs induce Bfc-mediated regulation of crq expression in macrophages remains unclear. Bfc regulates Crq expression and efferocytosis, but not macrophage development. Moreover, this study found that Bfc-mediated activation of crq transcription and Crq accumulation leads to positive feedback to promote increased Bfc expression, which is required for engulfment. As expected, the upregulation of Bfc expression occurred earlier than that of crq in S2 cells after incubation with ACs. Therefore, further studies are required to elucidate the upstream signals in the context of the crq-mediated regulation of bfc expression. As previous studies have shown that Crq is required for phagosome maturation during the clearance of neuronal debris by epithelial cells and bacterial clearance, further studies should be conducted to determine whether Bfc is involved in the clearance of neuronal debris (Zheng, 2021).

    This study is not without limitations. For instance, other potential regulators of efferocytosis, whose expression is not affected by ACs could not be detected in this study. In mammals, CD36 is involved in the clearance of ACs and regulates the host inflammatory response. As a CD36 family homolog, Crq promotes the clearance of ACs and bacterial uptake via efferocytosis. Researchers have reported that the GATA factor Srp is required for Crq expression; this study confirmed this finding and showed that Srp directly binds to the crq promoter via its GATA binding site, which is enhanced by Bfc. However, no apparent Bfc homologs exist in vertebrates, and whether GATA factors regulate the CD36 family in a mechanism similar to that in flies remains unclear. Nevertheless, it is predicted that one or more functional homologs of Bfc may exist in mammals and are likely involved in apoptotic cell clearance. Unraveling them as well as determining whether and how bfc participates in eliminating pathogens and innate immunity is essential (Zheng, 2021).

    In summary, this study has shown that the expression of the engulfment receptor Crq is transcriptionally regulated by the presence of ACs, via Srp, and its newly identified cofactor, Bfc. Altogether, these findings imply that macrophages adopt a precise mechanism to increase the expression of engulfment receptors to boost their phagocytic activity, in the presence of excessive ACs. A similar role and mechanism is anticipated in the context of mammalian engulfment receptors in response to excessive ACs. Therefore, the findings of this study have significant implications for a wide range of human diseases, including those associated with aberrant apoptotic cell death and efferocytosis, such as tumor progression, neurodegenerative disorders, and other severe inflammatory conditions (Zheng, 2021).

    Disruption of the lipolysis pathway results in stem cell death through a sterile immunity-like pathway in adult Drosophila

    Previous work has shown that the Arf1-mediated lipolysis pathway sustains stem cells and cancer stem cells (CSCs); its ablation resulted in necrosis of stem cells and CSCs, which further triggers a systemic antitumor immune response. This study shows that knocking down Arf1 in intestinal stem cells (ISCs) causes metabolic stress, which promotes the expression and translocation of ISC-produced damage-associated molecular patterns (DAMPs; Pretaporter [Prtp] and calreticulin [Calr]). DAMPs regulate macroglobulin complement-related (Mcr) expression and secretion. The secreted Mcr influences the expression and localization of enterocyte (EC)-produced Draper (Drpr) and LRP1 receptors (pattern recognition receptors [PRRs]) to activate autophagy in ECs for ATP production. The secreted ATP possibly feeds back to kill ISCs by activating inflammasome-like pyroptosis. This study identified an evolutionarily conserved pathway that sustains stem cells and CSCs, and its ablation results in an immunogenic cascade that promotes death of stem cells and CSCs as well as antitumor immunity (Aggarwal, 2022).

    Previous work showed that the Arf1-mediated lipolysis pathway is specifically activated in stem cells and sustains stem cells in adult Drosophila (Singh, 2016). Arf1 is one of the most evolutionarily conserved genes between Drosophila and mouse, with an amino acid identity of 95.6% between the two species. It was found recently that Arf1-mediated lipid metabolism sustains cancer stem cells (CSCs) and that its ablation triggers immunogenic-like death (immunogenic cell death [ICD]) of CSCs and induces antitumor immunity by exposing damage-associated molecular patterns (DAMPs; calreticulin [Calr], high-mobility group box 1 [HMGB1], and ATP) (Aggarwal, 2022).

    However, the molecular mechanism that coordinates stem cells/CSCs with neighboring cells to execute the biological processes (stem cell necrosis or anti-tumor immunity) is still unclear. This study dissected the molecular mechanism using the Drosophila genetic system. Knockdown of the pathway was found to promote stem cell death through an immunogenic-like and aging cascade. Ablation of Arf1-mediated lipid metabolism in Drosophila ISCs resulted in several aging-like hallmarks, including lipid droplet (LD) accumulation, Reactive oxygen species (ROS) accumulation, mitochondrial defects, mitophagy activation, and lysosomal protein aggregates, followed by an immunogenic-like cell death (Aggarwal, 2022).

    ICD is a process that releases DAMPs and activates immune responses to destroy damaged or stressed cells in the absence of microbial components. These molecules are often present in a given cell compartment and are not expressed or are only somewhat expressed under physiological conditions but strongly induced and then translocated to the cell surface or extracellular space under conditions of stress, damage, or injury. The most important DAMPs are (1) pre-apoptotic exposure of the ER-sessile molecular chaperone Calr on the cell surface, (2) release of the non-histone nuclear protein HMGB1 into the extracellular space, and (3) active secretion of ATP. With respect to tumors, the surface-exposed Calr facilitates engulfment of tumor-associated antigens by binding to LRP1/CD91 receptors (pattern recognition receptors [PRRs]) on dendritic cells (DCs). During ICD, Calr interacts with another protein, ERp57, and the two are rapidly translocated to the cell surface from the ER lumen before the cells exhibit any sign of apoptosis. ERp57 is a disulfide isomerase that has several thioredoxin-like domains and regulates cell redox homeostasis. Knocking down Arf1-mediated lipolysis in ISCs was found to promote the expression and translocation of ISC-produced DAMPs (Pretaporter [Prtp] and Calr). Like ERp57, Prtp is a disulfide isomerase with several thioredoxin-like domains. The DAMPs may then regulate the expression and secretion of the protein macroglobulin complement-related (Mcr; a complement C5 homolog). The secreted Mcr possibly further controls the expression and localization of EC-produced Draper [Drpr] and LRP1 receptors (PRRs) to activate autophagy in ECs for ATP production. The secreted ATP likely feeds back to kill ISCs by activating inflammasome-like pyroptosis. Therefore, Arf1-mediated lipid metabolism is crucial for stem cell maintenance, and its ablation promotes stem cell decay and anti-tumor immunity through an immunogenic aging cascade (Aggarwal, 2022).

    Stem cell functional decay or decline may be one of the important causes of organismal aging and disease. This study demonstrated that Arf1-mediated lipid metabolism sustains stem cells and that its ablation triggers an immunogenic-like stem cell death cascade. The dying stem cells display the following features: LD accumulation, mitochondrial defects, ROS production, ER stress and release of DAMPs to activate PRRs in neighboring ECs, mitophagy activation, lysosomal protein aggregations, and ISC necrosis through inflammasome-like pyroptosis. These features are similar to hallmarks of aging. Arf1 ablation in ISCs might trigger a stem cell aging and death cascade. The gold standard method for evaluating ICD is in vivo tumor vaccination. Previously an experiment of vaccination was performed in Arf1-ablated mice. The current study has demonstrated that many of the factors that contribute to ICD are expressed and function in Arf1-ablated flies, indicating that the pathway is partially conserved between Drosophila and mammals. However, it is important to confirm conserved biological functions of the ICD in Drosophila in future experiments. Similarly, inflammasome pyroptosis is only partially conserved between Drosophila and mammals. It is important to confirm the pathway by using inflammasome markers and demonstrate conserved biological functions of the pathway in Drosophila in future experiments (Aggarwal, 2022).

    A previous report demonstrated that Mcr, through Drpr, cell non-autonomously regulates autophagy during wound healing and salivary gland cell death in Drosophila and that Prtp is not involved in this Mcr-Drpr-mediated autophagy induction. Mcr is an analog of mammalian C1q/C5. C1q binds to the Calr-LRP1 coreceptor in mammals, and Mcr binds to LRP1 (Flybase) in Drosophila. This study found that Calr and Prtp function in parallel or downstream of the Arf1-lipolysis pathway and regulate the expression of Mcr and LRP1. Mcr and LRP1 further regulate each other and control the expression of Drpr. Calr and Prtp also regulate the expression of their respective receptors, LRP1 and Drpr. This information suggests that two interconnected complexes, Calr-Mcr-LRP1 and Prtp-Drpr, function downstream of the Arf1-lipolysis pathway and coordinately regulate ISC death (Aggarwal, 2022).

    In the mammalian immune system, DCs are activated after DAMPs bind to PRRs on their surface. The activated DCs present antigens to T cells, and the activated T cells kill damaged cells. The current study found that ablation of the COPI/Arf1-mediated lipolysis-β-oxidation pathway in stem cells induced expression of DAMPs, which then activate the phagocytic ECs through PRRs (LRP1 and Drpr) on the ECs to kill the stem cells. These findings suggest that such a coordinated cell death process is not limited to mammalian immune responses. In another naturally occurring example, Drpr pathway phagocytosis genes in follicle cells (FCs) non-autonomously promote nurse cell (NC) death in the developing Drosophila ovary. Although it is not clear how the stretch FCs time the precise developmental death of NCs, in light of the present findings, it is possible that a metabolic or stress signal during this developmental stage increases DAMPs in NCs to activate the Drpr pathway in FCs and non-autonomously promote NC death. DAMPs are also induced in organs during organ transplantation as a result of ischemic damage from the interrupted blood supply while the organ is outside of the body. The DAMPs induced in a graft stimulate immune responses mediated by host innate cells at the site of the graft and the donor's innate immune system and contribute to graft rejection. Drpr-mediated phagocytosis is also an essential process during development and in maintenance of tissue homeostasis in several systems. As mentioned above, the Mcr-Drpr pathway is involved in autophagy induction during wound healing and salivary gland cell death in Drosophila. It is proposed that such a coordinated cell death (CCD) is a novel and general cell death process in which death of abnormal or altered cells occurs by first sending danger signals (such as DAMPs) and then activating neighboring cells to execute the death process. The abnormality or alteration can be metabolic stress (such as disruption of Arf1-mediated lipid metabolism in stem cells), developmental changes (such as NC death during Drosophila ovary development or salivary gland cell death during metamorphosis), or damage during wound healing or circulation blockage during ischemic damage or pathogen infection. The danger signals then activate phagocytes and other cells (such as T cells) to cell non-autonomously promote targeted cell death. CCD may mediate cell aging/death and organ degeneration under physiological conditions or CSC death and anti-tumor immunity under pathological conditions (Aggarwal, 2022).

    The finding that the DAMP-Mcr-LRP1/Drpr pathway connects metabolically stressed stem cells after Arf1 ablation to activation of phagocytic ECs to kill the stem cells will enable further dissection of the CCD mechanism in Drosophila. Arf1 is one of the most evolutionarily conserved genes, and the DAMP-Mcr/C1q-LRP1/Drpr pathway is well conserved throughout evolution. CCD involves coordination or communication of two or more different cells. Model organisms such as Drosophila, with their advanced genetic tractability and well-characterized cellular histology, will serve as valuable in vivo models for dissecting the detailed cellular and molecular mechanisms of CCD. These findings may lead to new therapeutic strategies for many human diseases, such as induction of anti-tumor immunity in individuals with cancer and the blocking of neuronal death in individuals with neurodegenerative conditions (Aggarwal, 2022).

    This study has identified an evolutionarily conserved pathway that sustains stem cells, and its ablation results in an ICD cascade that promotes death of stem cells through inflammasome-like pyroptosis. It was demonstrated that many of the factors that contribute to ICD and inflammasome-like pyroptosis are expressed and function in Arf1-ablated flies. However, the gold standard method for evaluating ICD is in vivo tumor vaccination. The components of ICD and inflammasome-like pyroptosis are only partially conserved between Drosophila and mammals. It is important to further confirm the pathway by using inflammasome markers and demonstrate conserved biological functions of the pathway in Drosophila in future experiments (Aggarwal, 2022).

    Actin remodeling mediates ROS production and JNK activation to drive apoptosis-induced proliferation

    Stress-induced cell death, mainly apoptosis, and its subsequent tissue repair is interlinked although knowledge of this connection is still very limited. An intriguing finding is apoptosis-induced proliferation (AiP), an evolutionary conserved mechanism employed by apoptotic cells to trigger compensatory proliferation of their neighboring cells. Studies using Drosophila as a model organism have revealed that apoptotic caspases and c-Jun N-terminal kinase (JNK) signaling play critical roles to activate AiP. For example, the initiator caspase Dronc, the caspase-9 ortholog in Drosophila, promotes activation of JNK leading to release of mitogenic signals and AiP. Recent studies further revealed that Dronc relocates to the cell cortex via Myo1D, an unconventional myosin, and stimulates production of reactive oxygen species (ROS) to trigger AiP. During this process, ROS can attract hemocytes, the Drosophila macrophages, which further amplify JNK signaling cell non-autonomously. However, the intrinsic components connecting Dronc, ROS and JNK within the stressed signal-producing cells remain elusive. This study identified LIM domain kinase 1 (LIMK1), a kinase promoting cellular F-actin polymerization, as a novel regulator of AiP. F-actin accumulates in a Dronc-dependent manner in response to apoptotic stress. Suppression of F-actin polymerization in stressed cells by knocking down LIMK1 or expressing Cofilin, an inhibitor of F-actin elongation, blocks ROS production and JNK activation, hence AiP. Furthermore, Dronc and LIMK1 genetically interact. Co-expression of Dronc and LIMK1 drives F-actin accumulation, ROS production and JNK activation. Interestingly, these synergistic effects between Dronc and LIMK1 depend on Myo1D. Therefore, F-actin remodeling plays an important role mediating caspase-driven ROS production and JNK activation in the process of AiP (Farrell, 2022).

    E2F1 promotes, JNK and DIAP1 inhibit, and chromosomal position has little effect on radiation-induced Loss of Heterozygosity in Drosophila

    Loss of Heterozygosity (LOH) can occur when a heterozygous mutant cell loses the remaining wild type allele to become a homozygous mutant. LOH can have physiological consequences if, for example, the affected gene encodes a tumor suppressor. This study used two fluorescent reporters to study mechanisms of LOH induction by X-rays, a type of ionizing radiation (IR), in Drosophila larval wing discs. IR is used to treat more than half of cancer patients, so understanding its effects is of biomedical relevance. IR-induced LOH does not correlate with the chromosomal position of the LOH locus, unlike previously shown for spontaneously occurring LOH. Like spontaneous LOH, however, IR-induced LOH of X-linked loci shows a sex-dependence, occurring predominately in females. A focused genetic screen identified E2F1 as a key promotor of LOH and further testing suggests a mechanism involving its role in cell cycle regulation rather than apoptosis. The QF/QS LOH reporter was combined with QUAS-transgenes to manipulate gene function after LOH induction. This approach identified JNK signaling and apoptosis as key determinants of LOH maintenance. These studies reveal previously unknown mechanisms for generation and maintenance of cells with chromosome aberrations after exposure to IR (Brown, 2023).

    Spoonbill positively regulates JNK signalling mediated apoptosis in Drosophila melanogaster

    Spoonbill (Spoon) is a putative A-kinase anchoring protein in Drosophila. This report has unravelled a novel function of Spoon protein in the regulation of the apoptotic pathway. The Drosophila TNFα homolog, Eiger, induces apoptosis via activation of the JNK pathway. This study shows that Spoonbill is a positive regulator of the Eiger-induced JNK signalling. Further genetic interaction studies show that spoon interacts with components of the JNK pathway, TGF-β activated kinase 1 (Tak1 - JNKKK), hemipterous (hep - JNKK) and basket (bsk - JNK). Interestingly, Spoonbill alone can also induce ectopic activation of the JNK pathway in a context-specific manner. To understand the molecular mechanism underlying Spoonbill-mediated modulation of the JNK pathway, the interaction between Spoon and Drosophila JNK was assessed. basket encodes the only known JNK in Drosophila. This serine/threonine-protein kinase phosphorylates Jra/Kay, which transcriptionally regulate downstream targets like Matrix metalloproteinase 1 (Mmp1), puckered (puc), and proapoptotic genes hid, reaper and grim. Interestingly, it was found that Spoonbill colocalises and co-immunoprecipitates with the Basket protein in the developing photoreceptor neurons. It is proposed that Spoon plays a vital role in JNK-induced apoptosis. Furthermore, stress-induced JNK activation underlying Parkinson's Disease was also examined. In the Parkinson's Drosophila model of neurodegeneration, depletion of Spoonbill leads to a partial reduction of JNK pathway activation, along with improvement in adult motor activity. These observations suggest that the putative scaffold protein Spoonbill is a functional and physical interacting partner of the Drosophila JNK protein, Basket. Spoon protein is localised on the outer mitochondrial membrane (OMM), which may perhaps provide a suitable subcellular niche for activation of Drosophila Basket protein by its kinases which induce apoptosis (Dos, 2023).

    Bilateral JNK activation is a hallmark of interface surveillance and promotes elimination of aberrant cells
    Tissue-intrinsic defense mechanisms eliminate aberrant cells from epithelia and thereby maintain the health of developing tissues or adult organisms. 'Interface surveillance' comprises one such distinct mechanism that specifically guards against aberrant cells which undergo inappropriate cell fate and differentiation programs. The cellular mechanisms which facilitate detection and elimination of these aberrant cells are currently unknown. This study findd that in Drosophila imaginal discs, clones of cells with inappropriate activation of cell fate programs induce bilateral JNK activation at clonal interfaces, where wild type and aberrant cells make contact. JNK activation is required to drive apoptotic elimination of interface cells. Importantly, JNK activity and apoptosis are highest in interface cells within small aberrant clones, which likely supports the successful elimination of aberrant cells when they arise. These findings are consistent with a model where clone size affects the topology of interface contacts and thereby the strength of JNK activation in wild type and aberrant interface cells. Bilateral JNK activation is unique to 'interface surveillance' and is not observed in other tissue-intrinsic defense mechanisms, such as classical 'cell-cell competition'. Thus, bilateral JNK interface signaling provides an independent tissue-level mechanism to eliminate cells with inappropriate developmental fate but normal cellular fitness. Finally, oncogenic Ras-expressing clones activate 'interface surveillance' but evade elimination by bilateral JNK activation. Combined, this work establishes bilateral JNK interface signaling and interface apoptosis as a new hallmark of interface surveillance and highlights how oncogenic mutations evade tumor suppressor function encoded by this tissue-intrinsic surveillance system (Prasad, 2023).

    Mutation and apoptosis are well-coordinated for protecting against DNA damage-inducing toxicity in Drosophila

    No reports have comprehensively explored the direct relationship between apoptosis and somatic cell mutations induced by various mutagenic factors. Mutation was examined by the wing-spot test, which is used to detect somatic cell mutations, including chromosomal recombination. Apoptosis was observed in the wing discs by acridine orange staining in situ. After treatment with chemical mutagens, ultraviolet light (UV), and X-ray, both the apoptotic frequency and mutagenic activity increased in a dose-dependent manner at non-toxic doses. When DNA repair-deficient Drosophila strains were used, the correlation coefficient of the relationship between apoptosis and mutagenicity, differed from that of the wild-type. To explore how apoptosis affects the behavior of mutated cells, the spot size was determined, i.e., the number of mutated cells in a spot. In parallel with an increase in apoptosis, the spot size increased with MNU or X-ray treatment dose-dependently; however, this increase was not seen with UV irradiation. In addition, BrdU incorporation, an indicator of cell proliferation, in the wing discs was suppressed at 6 h, with peak at 12 h post-treatment with X-ray, and that it started to increase again at 24 h; however, this was not seen with UV irradiation. It is concluded that damage-induced apoptosis and mutation might be coordinated with each other, and the frequency of apoptosis and mutagenicity are balanced depending on the type of DNA damage. From the data of the spot size and BrdU incorporation, it is possible that mutated cells replace apoptotic cells due to their high frequency of cell division, resulting in enlargement of the spot size after MNU or X-ray treatment. It is consider that the induction of mutation, apoptosis, and/or cell growth varies in multi-cellular organisms depending on the type of the mutagens, and that their balance and coordination have an important function to counter DNA damage for the survival of the organism (Toyoshima-Sasatani, 2023).

    Epidermal growth factor receptor signaling protects epithelia from morphogenetic instability and tissue damage in Drosophila

    Dying cells in the epithelia communicate with neighboring cells to initiate coordinated cell removal to maintain epithelial integrity. Naturally occurring apoptotic cells are mostly extruded basally and engulfed by macrophages. This study has investigated the role of Epidermal growth factor (EGF) receptor (EGFR) signaling in the maintenance of epithelial homeostasis. In Drosophila embryos, epithelial tissues undergoing groove formation preferentially enhanced extracellular signal-regulated kinase (ERK) signaling. In EGFR mutant embryos at stage 11, sporadic apical cell extrusion in the head initiates a cascade of apical extrusions of apoptotic and non-apoptotic cells that sweeps the entire ventral body wall. This study shows that this process is apoptosis dependent, and clustered apoptosis, groove formation, and wounding sensitize EGFR mutant epithelia to initiate massive tissue disintegration. It was further shown that tissue detachment from the vitelline membrane, which frequently occurs during morphogenetic processes, is a key trigger for the EGFR mutant phenotype. These findings indicate that, in addition to cell survival, EGFR plays a role in maintaining epithelial integrity, which is essential for protecting tissues from transient instability caused by morphogenetic movement and damage (Yoshida, 2023).

    Toll-9 interacts with Toll-1 to mediate a feedback loop during apoptosis-induced proliferation in Drosophila

    Drosophila Toll-1 and all mammalian Toll-like receptors regulate innate immunity. However, the functions of the remaining eight Toll-related proteins in Drosophila are not fully understood. This study shows that Drosophila Toll-9 is necessary and sufficient for a special form of compensatory proliferation after apoptotic cell loss (undead apoptosis-induced proliferation [AiP]). Mechanistically, for AiP, Toll-9 interacts with Toll-1 to activate the intracellular Toll-1 pathway for nuclear translocation of the NF-κB-like transcription factor Dorsal, which induces expression of the pro-apoptotic genes reaper and hid. This activity contributes to the feedback amplification loop that operates in undead cells. Given that Toll-9 also functions in loser cells during cell competition, this study defines a general role of Toll-9 in cellular stress situations leading to the expression of pro-apoptotic genes that trigger apoptosis and apoptosis-induced processes such as AiP. This work identifies conceptual similarities between cell competition and AiP (Shields, 2022).

    Since the discovery of the original Toll gene in Drosophila (Toll-1 hereafter), a large number of Toll-related genes have been identified in both insects and mammals. The Drosophila genome encodes a total of 9 Toll-related genes, including Toll-1, while mammalian genomes encode between 10 and 13 Toll-like receptors (TLRs). TLRs are single-pass transmembrane proteins that upon ligand stimulation usually trigger a conserved intracellular signaling pathway, culminating in the activation of NF-κB transcription factors. Initially identified as an essential gene for dorsoventral patterning in the early Drosophila embryo, Toll-1 was later also found to be a critical component for innate immunity. In this function, Toll-1 signaling via the NF-κB transcription factors Dorsal and Dorsal-related immunity factor (Dif) induces the expression of anti-microbial peptides (AMPs) that mediate innate immunity. A role in innate immunity has also been demonstrated for all mammalian TLRs. Likewise, Drosophila Toll-8 (aka Tollo) regulates immunity in the trachea. Toll-7 may regulate anti-viral responses, albeit through an NF-κB-independent mechanism. However, for the remaining Toll-related proteins in Drosophila, a function in innate immunity has not been clearly demonstrated (Shields, 2022).

    Of particular interest is Toll-9 in Drosophila because it behaves genetically most similar to Toll-1, and its intracellular TIR domain is most closely related to those of the mammalian TLRs. Overexpression of Toll-9 results in the production of AMPs, which led to the proposal that Toll-9 might be involved in innate immunity. However, a loss-of-function analysis with a defined Toll-9 null allele did not confirm this prediction. Nevertheless, although Drosophila Toll-9 does not appear to be directly involved in regulating expression of AMPs, it has been implicated together with a few other Toll-related proteins in cell competition, an organismal surveillance program that monitors cellular fitness and eliminates cells of reduced fitness (losers). Depending on the type of cell competition, Toll-9 participates in the expression of the pro-apoptotic genes reaper and hid in loser cells, triggering their elimination . Therefore, it has been proposed that the function of Toll signaling for elimination of bacterial pathogens by AMPs and for elimination of unfit cells by pro-apoptotic genes bears a conceptual resemblance between innate immunity and cell competition (Shields, 2022).

    Apoptosis is an evolutionarily well-conserved process of cellular suicide mediated by a highly specialized class of Cys-proteases, termed caspases. In Drosophila, the pro-apoptotic genes reaper and hid promote the activation of the initiator caspase Dronc (caspase-9 homolog in Drosophila). Dronc activates the effector caspases DrICE and Dcp-1 (caspase-3 and caspase-7 homologs), which trigger the death of the cell. However, caspases not only induce apoptosis but can also have non-apoptotic functions such as apoptosis-induced proliferation (AiP), during which caspases in apoptotic cells promote the proliferation of surviving cells independently of their role in apoptosis. Early work has shown that AiP requires the initiator caspase Dronc (Shields, 2022).

    To reveal the mechanism of AiP, this study expressedthe pro-apoptotic gene hid and the apoptosis inhibitor p35 simultaneously using the ey-Gal4 driver (referred to as ey > hid,p35) which drives expression in the larval eye disc anterior to the morphogenetic furrow (MF) . p35 encodes a specific inhibitor of the effector caspases DrICE and Dcp-1 but does not block the activity of Dronc. In ey > hid,p35-expressing discs, the apoptotic pathway is activated by hid expression but blocked downstream because of DrICE and Dcp-1 inhibition by P35, rendering cells in an 'undead' condition. Although apoptosis is inhibited in undead tissue, AiP still occurs because of non-apoptotic signaling by the initiator caspase Dronc, triggering hyperplasia of the anterior portion of the larval eye imaginal discs at the expense of the posterior eye field, which is reduced in size. Combined, these effects result in overgrowth of the adult head capsule, but a reduction or even absence of the eyes in the adult animal (Shields, 2022).

    Using the undead model, this study showed that AiP is mediated by extracellular reactive oxygen species (ROS) generated by the NADPH oxidase Duox. ROS trigger the recruitment of hemocytes, Drosophila immune cells most similar to mammalian macrophages, to undead imaginal discs. Hemocytes in turn release signals that stimulate JNK activity in undead cells, which then promotes AiP. In addition, JNK can also induce the expression of hid, thus setting up an amplification loop in undead cells which continuously signals for AiP (Shields, 2022).

    Given that undead cells are abnormal cells with potentially altered cellular fitness and that signaling by Toll-related proteins surveilles cellular fitness, this study examined if signaling by Toll-9 has an important function for undead AiP. This study shows that Toll-9 is strongly up-regulated in undead cells and is necessary for the overgrowth of undead tissue. Overexpression of Toll-9 with p35 induces all hallmarks of undead AiP signaling, including Duox-dependent ROS generation, hemocyte recruitment and JNK signaling. Mechanistically, genetic evidence is provided for a heterologous interaction between Toll-9 and Toll-1, which engages the canonical Toll-1 signaling pathway to promote nuclear translocation of the NF-κB-like transcription factor Dorsal, which induces the expression of reaper and hid. This activity contributes to the establishment of the feedback amplification loop that signals continuously for AiP. In conclusion, although Toll-9 does not appear to have an important function in innate immunity, it appears to be involved in the expression of pro-apoptotic genes such as reaper and hid in stress situations such as cell competition and undead AiP (Shields, 2022).

    Toll-9 is the most closely related Drosophila TLR compared with mammalian TLRs, but a biological function of Toll-9 has not been clearly defined. All mammalian TLRs are involved in innate immunity; therefore, the close homology has led to the prediction that Drosophila Toll-9 also participates in innate immunity. However, in Toll-9 mutants, the basal as well as bacterially induced AMP production is not affected, leading to the conclusion that Toll-9 is not involved in innate immunity (Narbonne-Reveau, 2011). Nevertheless, instead of eliminating foreign pathogens, previous work has shown that Toll-9 together with several other Toll-related receptors participates in elimination of unfit cells during cell competition. This is achieved through the expression of the pro-apoptotic gene rpr. This study demonstrates that Toll-9 has a similar rpr- and hid-inducing function during AiP, thereby adding to the database of Toll-9 function (Shields, 2022).

    To identify the mechanism by which Toll-9 participates in AiP, advantage was taken of the observation that misexpression of Toll-9 is sufficient to induce overgrowth of ey > p35 animals. There are multiple aspects of this phenotype that are worth being discussed. First, key for many of the observations presented in this paper is the presence of P35, a very efficient inhibitor of the effector caspases DrICE and Dcp-1. In the absence of P35, overexpression of Toll-9 does not induce any obvious phenotypes in eye discs or adult heads. The exact reason for this P35 dependence is currently unknown, but it has also been observed upon misexpression of other genes involved in AiP, such as Myo1D, Toll-1, and SpzAct. The only known function of P35 is to inhibit DrICE and Dcp-1. Therefore, one possible explanation for the P35 dependence is that these caspases cleave and inactivate an as yet unidentified component of the AiP network, possibly to block inappropriate AiP under normal conditions. In the presence of P35, the AiP-blocking activity of DrICE is inhibited and with the addition of an AiP-inducing stimulus such as misexpression of Toll-9, AiP is engaged and can trigger tissue overgrowth. Second, the data show that ectopic p35,Toll-9 co-expression triggers overgrowth through a similar mechanism as hid,p35 co-expression. This includes Dronc activation, Duox-generated ROS, hemocyte recruitment, and JNK activation. These similarities allow placing the function of Toll-9 into the AiP network (Shields, 2022).

    Third, mis-expressed Toll-9 can genetically interact with Toll-1. This interaction results in nuclear translocation of Dorsal and is dependent on Myd88, Tube, and Pelle, all canonical components of the intracellular Toll-1 signaling pathway. Importantly, the nuclear translocation of Dorsal and the p35,Toll-9-induced overgrowth is also dependent on Toll-1, suggesting that the activation of the Myd88/Tube/Pelle pathway is directly triggered by Toll-1 and not by Toll-9. Mechanistically, Toll-9 may activate Toll-1 either directly through hetero-dimerization or mediated by additional factors. Future work will be necessary to identify the molecular mechanism of the Toll-9/Toll-1 interaction (Shields, 2022).

    Fourth, the outcome of the Toll-9/Toll-1 interaction is the expression of the pro-apoptotic genes reaper and hid. Because Toll-9 expression is strongly up-regulated in undead cells, these data suggest that Toll-9-induced expression of reaper and hid in undead cells is setting up an amplification loop. The cause of the strong transcriptional upregulation of Toll-9 in undead cells is unknown, but it requires JNK activity. The Toll-9 amplification loop contributes to the strength of undead signaling during AiP and propels the overgrowth of the tissue (Shields, 2022).

    Fifth, another important question is how Toll signaling becomes activated during AiP. Toll-1 is activated by the ligand Spatzle during embryogenesis and the immune response. Spz requires proteolytic processing for activation, which during the immune response is mediated by the Ser-protease Spatzle-processing enzyme (SPE). Consistently, SPE RNAi can suppress both the ey > p35,Toll-9INTRA and ey > hid,p35-induced overgrowth phenotypes. SPE RNAi suppressed ey > p35,Toll-9INTRA, which lacks the extracellular domain and should be insensitive to a ligand. Thus, the suppression of ey > p35,Toll-9INTRA suggests that SPE does not act through Toll-9 but instead on Toll-1. This result is consistent with a recent finding that Toll-9 RNAi cannot suppress apoptosis induced by a dominant active SPE (SPEAct) transgene. Although that there is an unknown Toll-9 ligand cannot be ruled out, Toll-9 may not need to be activated by a ligand. Toll-9 naturally carries an amino acid substitution in the cysteine-rich extracellular domain similar to the gain-of-function Toll-11 mutant. Indeed, Toll-9 behaves as a constitutively active receptor in cell culture assays. As TLRs can form homo- and heterodimers, it is possible that the constitutive activity of Toll-9 and the strong transcriptional upregulation of Toll-9 together with ligand stimulation of Toll-1 by Spz is sufficient for the activation of the Toll-1/Toll-9 complex. However, it remains unknown how SPE becomes activated during AiP (Shields, 2022).

    With these considerations in mind, the following model for Toll-9 function during undead AiP emerges. The initial stimulus for AiP is the Gal4-induced expression of hid and p35, which leads to the activation of Dronc. Because of P35, Dronc cannot induce apoptosis but instead activates Duox for generation of ROS. ROS attract hemocytes which release signals for JNK activation in undead cells. JNK signaling directly or indirectly induces Toll-9 transcription. Up-regulated Toll-9 interacts with Toll-1, and after Spz ligation the Myd88/Tube/Pelle pathway triggers the nuclear accumulation of Dorsal and potentially Dif. These factors transcriptionally induce reaper and hid expression setting up the feedback amplification loop, which maintains and propels AiP and overgrowth (Shields, 2022).

    One other interesting question to examine in the future will be how the intracellular pathway of Toll-1 signaling including Dorsal and Dif can induce different target genes under different conditions. For dorsoventral patterning of the Drosophila embryo, Dorsal induces the expression of twist and snail). During immune responses in the fat body, it promotes the expression of AMP genes as well as Kennedy pathway genes for the synthesis of phospholipids, while during cell competition and AiP which occur in larval imaginal discs, pro-apoptotic genes hid and rpr are induced. One potential answer to this question is that the specificity of Toll-1 signaling may be modified by the interaction with Toll-9. Although this interaction occurs at the plasma membrane, it also might influence the activity in the nucleus. It will also be interesting to examine if Toll-1 can interact with some or all of the other Toll-related receptors in Drosophila and how this interaction might influence the specificity of the transcriptional outcome. Although Toll-9 in Drosophila does not appear to be required for innate immunity, on the basis of its non-essential function to induce AMP production during bacterial infection (Narbonne-Reveau, 2011), the currenrt work and work by others (Meyer, 2014) reveals that Toll-9 may have a function during stress responses which involves expression of pro-apoptotic genes such as rpr and hid. That was demonstrated previously for cell competition and now for undead AiP, indicating potential similarities between cell competition and undead AiP. At first, such similarities appear to be at odds with the common dogmas that proliferating winner cells trigger apoptosis of loser cells, while during AiP, apoptotic cells induce proliferation of surviving cells. However, it has also been reported that loser cells have a much more active role during cell competition and can promote the winner status of cells with increased fitness. Therefore, there appear to be significant similarities between cell competition and undead AiP. The common denominator for both systems is the expression of pro-apoptotic genes. These responses have different outcomes in both systems. During cell competition, this response involves the death of the loser cells. During undead AiP, it sets up the amplification loop known to operate in undead cells, which propels hyperplasia and tissue overgrowth (Shields, 2022).

    This work was performed largely under undead conditions (i.e., in the presence of the effector caspase inhibitor p35, which is not an endogenous gene in Drosophila). In reality, however, in the absence of p35, effector caspases are also activated in apoptotic cells, which will eventually lead to the death of the cell. Therefore, the question arises as to how apoptosis and AiP are linked to allow compensatory proliferation under normal conditions. Recently, evidence has been presented that certain apoptotic cells (dying enterocytes in the adult intestine) can adopt a transiently undead-like state that enables them to signal for AiP before they are dying and removed. The transiently undead-like state is achieved by transient localization of Dronc to the plasma membrane, which might serve as a non-apoptotic compartment of the cell. In that way, apoptotic cells, before they die, can trigger AiP in a p35-independent manner. Therefore, in future research, it will be important to examine if the Toll-9/Toll-1 interaction sets up a similar amplification loop in transiently undead enterocytes (Shields, 2022).

    Integrins Cooperate With the EGFR/Ras Pathway to Preserve Epithelia Survival and Architecture in Development and Oncogenesis

    Adhesion to the extracellular matrix (ECM) is required for normal epithelial cell survival. Disruption of this interaction leads to a specific type of apoptosis known as anoikis. Yet, there are physiological and pathological situations in which cells not connected to the ECM are protected from anoikis, such as during cell migration or metastasis. The main receptors transmitting signals from the ECM are members of the integrin family. However, although integrin-mediated cell-ECM anchorage has been long recognized as crucial for epithelial cell survival, the in vivo significance of this interaction remains to be weighed. This study used the Drosophila wing imaginal disc epithelium to analyze the importance of integrins as survival factors during epithelia morphogenesis. Reducing integrin expression in the wing disc induces caspase-dependent cell death and basal extrusion of the dead cells. In this case, anoikis is mediated by the activation of the JNK pathway, which in turn triggers expression of the proapoptotic protein Hid. In addition, the results strongly suggest that, during wing disc morphogenesis, the EGFR pathway protects cells undergoing cell shape changes upon ECM detachment from anoikis. Furthermore, it was shown that oncogenic activation of the EGFR/Ras pathway in integrin mutant cells rescues them from apoptosis while promoting their extrusion from the epithelium. Altogether, these results support the idea that integrins promote cell survival during normal tissue morphogenesis and prevent the extrusion of transformed cells (Valencia-Exposito, 2022).

    Anastasis Drives Senescence and Non-Cell Autonomous Neurodegeneration in the Astrogliopathy Alexander Disease

    Anastasis is a recently described process in which cells recover after late-stage apoptosis activation. The functional consequences of anastasis for cells and tissues are not clearly understood. Using Drosophila, rat and human cells and tissues, including analyses of both males and females, this study presents evidence that glia undergoing anastasis in the primary astrogliopathy Alexander disease subsequently express hallmarks of senescence. These senescent glia promote non-cell autonomous death of neurons by secreting interleukin family cytokines. These findings demonstrate that anastasis can be dysfunctional in neurologic disease by inducing a toxic senescent population of astroglia (Wang, 2022).

    Autophagy impairment and lifespan reduction caused by Atg1 RNAi or Atg18 RNAi expression in adult fruit flies (Drosophila melanogaster

    Autophagy, an autophagosome and lysosome-based eukaryotic cellular degradation system, has previously been implicated in lifespan regulation in different animal models. This report shows that expression of the RNAi transgenes targeting the transcripts of the key autophagy genes Atg1 or Atg18 in adult fly muscle or glia does not affect the overall levels of autophagosomes in those tissues and does not change the lifespan of the tested flies, but lifespan reduction phenotype has become apparent when Atg1 RNAi or Atg18 RNAi is expressed ubiquitously in adult flies or after autophagy is eradicated through the knockdown of Atg1 or Atg18 in adult fly adipocytes. Lifespan reduction was also observed when Atg1 or Atg18 was knocked down in adult fly enteroblasts and midgut stem cells. Over-expression of wild type Atg1 in adult fly muscle or adipocytes reduces lifespan and causes accumulation of high levels of ubiquitinated protein aggregates in muscles. These research data have highlighted the important functions of the key autophagy genes in adult fly adipocytes, enteroblasts, and midgut stem cells and their undetermined roles in adult fly muscle and glia for lifespan regulation (Bierlein, 2023).

    Neuronal atg1 Coordinates Autophagy Induction and Physiological Adaptations to Balance mTORC1 Signalling

    The mTORC1 nutrient-sensing pathway integrates metabolic and endocrine signals into the brain to evoke physiological responses to food deprivation, such as autophagy. Nevertheless, the impact of neuronal mTORC1 activity on neuronal circuits and organismal metabolism remains obscure. This study shows that mTORC1 inhibition acutely perturbs serotonergic neurotransmission via proteostatic alterations evoked by the autophagy inducer Atg1. Neuronal ATG1 alters the intracellular localization of the serotonin transporter, which increases the extracellular serotonin and stimulates the 5HTR7 postsynaptic receptor. 5HTR7 enhances food-searching behaviour and ecdysone-induced catabolism in Drosophila. Along similar lines, the pharmacological inhibition of mTORC1 in zebrafish also stimulates food-searching behaviour via serotonergic activity. These effects occur in parallel with neuronal autophagy induction, irrespective of the autophagic activity and the protein synthesis reduction. In addition, ectopic neuronal atg1 expression enhances catabolism via insulin pathway downregulation, impedes peptidergic secretion, and activates non-cell autonomous cAMP/PKA. The above exert diverse systemic effects on organismal metabolism, development, melanisation, and longevity. It is concluded that neuronal atg1 aligns neuronal autophagy induction with distinct physiological modulations, to orchestrate a coordinated physiological response against reduced mTORC1 activity (Metaxakis, 2023).

    Polyploidy-associated autophagy promotes larval tracheal histolysis at Drosophila metamorphosis

    Polyploidy is an extended phenomenon in biology. However, its physiological significance and whether it defines specific cell behaviors is not well understood. Polyploidy connection to macroautophagy/autophagy was examined, using the larval respiratory system of Drosophila as a model. This system comprises cells with the same function yet with notably different ploidy status, namely diploid progenitors and their polyploid larval counterparts, the latter destined to die during metamorphosis. An association was identified between polyploidy and autophagy and higher endoreplication status was found to correlate with elevated autophagy. Finally, it is reported that tissue histolysis in the trachea during Drosophila metamorphosis is mediated by autophagy, which triggers the apoptosis of polyploid cells (Pino-Jimenez, 2023).

    Dysfunction of lipid storage droplet-2 suppresses endoreplication and induces JNK pathway-mediated apoptotic cell death in Drosophila salivary glands

    The lipid storage droplet-2 (LSD-2) protein of Drosophila is a homolog of mammalian perilipin 2, which is essential for promoting lipid accumulation and lipid droplet formation. The function of LSD-2 as a regulator of lipolysis has also been demonstrated. However, other LSD-2 functions remain unclear. To investigate the role of LSD-2, tissue-specific depletion in the salivary glands of Drosophila was performed using a combination of the Gal4-upstream activating sequence system and RNA interference. LSD-2 depletion inhibited the entry of salivary gland cells into the endoreplication cycle and delayed this process by enhancing CycE expression, disrupting the development of this organ. The deficiency of LSD-2 expression enhanced reactive oxygen species production in the salivary gland and promoted JNK-dependent apoptosis by suppressing dMyc expression. This phenomenon did not result from lipolysis. Therefore, LSD-2 is vital for endoreplication cell cycle and cell death programs (Binh, 2022).

    Non-autonomous cell death induced by the Draper phagocytosis receptor requires signaling through the JNK and SRC pathways

    The last step of cell death is cell clearance, a process critical for tissue homeostasis. For efficient cell clearance to occur, phagocytes and dead cells need to reciprocally signal to each other. One important phenomenon that is under-investigated, however, is that phagocytes not only engulf corpses but contribute to cell death progression. The aims of this study were to determine how the phagocytic receptor Draper non-autonomously induces cell death, using the Drosophila ovary as a model system. Draper, expressed in epithelial follicle cells, was shown to require its intracellular signaling domain to kill the adjacent nurse cell population. Kinases Src42A, Shark and JNK (Bsk) were required for Draper-induced nurse cell death. Signs of nurse cell death occurred prior to apparent engulfment and required the caspase Dcp-1, indicating that it uses a similar apoptotic pathway to starvation-induced cell death. These findings indicate that active signaling by Draper is required to kill nurse cells via the caspase Dcp-1, providing novel insights into mechanisms of phagoptosis driven by non-professional phagocytes (Serizier, 2023).

    Lack of apoptosis leads to cellular senescence and tumorigenesis in Drosophila epithelial cells

    Programmed cell death (apoptosis) is a homeostasis program of animal tissues designed to remove cells that are unwanted or are damaged by physiological insults. To assess the functional role of apoptosis, the consequences were studied of subjecting Drosophila epithelial cells defective in apoptosis to stress or genetic perturbations that normally cause massive cell death. Many of those cells acquire persistent activity of the JNK pathway, which drives them into senescent status, characterized by arrest of cell division, cell hypertrophy, Senescent Associated β-gal activity (SA-β-gal), reactive oxygen species (ROS) production, Senescent Associated Secretory Phenotype (SASP) and migratory behaviour. Two classes of senescent cells were identified in the wing disc: 1) those that localize to the appendage part of the disc, express the upd, wg and dpp signalling genes and generate tumour overgrowths, and 2) those located in the thoracic region do not express wg and dpp nor they induce tumour overgrowths. Whether to become tumorigenic or non-tumorigenic depends on the original identity of the cell prior to the transformation. The p53 gene was also found to contribute to senescence by enhancing the activity of JNK (Garcia-Arias, 2023).

    Apoptotic extracellular vesicle formation via local phosphatidylserine exposure drives efficient cell extrusion

    Cell extrusion is a universal mode of cell removal from tissues, and it plays an important role in regulating cell numbers and eliminating unwanted cells. However, the underlying mechanisms of cell delamination from the cell layer are unclear. This study reports a conserved execution mechanism of apoptotic cell extrusion. Extracellular vesicle (EV) formation in extruding mammalian and Drosophila cells was found at a site opposite to the extrusion direction. Lipid-scramblase-mediated local exposure of phosphatidylserine is responsible for EV formation and is crucial for executing cell extrusion. Inhibition of this process disrupts prompt cell delamination and tissue homeostasis. Although the EV has hallmarks of an apoptotic body, its formation is governed by the mechanism of microvesicle formation. Experimental and mathematical modeling analysis illustrated that EV formation promotes neighboring cells' invasion. This study showed that membrane dynamics play a crucial role in cell exit by connecting the actions of the extruding cell and neighboring cells (Kira, 2023).

    Necrosis-induced apoptosis promotes regeneration in Drosophila wing imaginal discs

    Regeneration is a complex process that requires a coordinated genetic response to tissue loss. Signals from dying cells are crucial to this process and are best understood in the context of regeneration following programmed cell death, like apoptosis. Conversely, regeneration following unregulated forms of death, such as necrosis, have yet to be fully explored. This study has developed a method to investigate regeneration following necrosis using the Drosophila wing imaginal disc. Necrosis is shown to stimulate regeneration at an equivalent level to that of apoptosis-mediated cell death and activates a similar response at the wound edge involving localized JNK signaling. Unexpectedly, however, necrosis also results in significant apoptosis far from the site of ablation, which this study terms necrosis-induced apoptosis (NiA). This apoptosis occurs independent of changes at the wound edge and importantly does not rely on JNK signaling. Furthermore, it was found that blocking NiA limits proliferation and subsequently inhibits regeneration, suggesting that tissues damaged by necrosis can activate programmed cell death at a distance from the injury to promote regeneration (Klemm, 2021).

    Caspases maintain tissue integrity by an apoptosis-independent inhibition of cell migration and invasion

    Maintenance of tissue integrity during development and homeostasis requires the precise coordination of several cell-based processes, including cell death. In animals, the majority of such cell death occurs by apoptosis, a process mediated by caspase proteases. To elucidate the role of caspases in tissue integrity, this study investigated the behavior of Drosophila epithelial cells that are severely compromised for caspase activity. These cells acquire migratory and invasive capacities, either within 1-2 days following irradiation or spontaneously during development. Importantly, low levels of effector caspase activity, which are far below the threshold required to induce apoptosis, can potently inhibit this process, as well as a distinct, developmental paradigm of primordial germ cell migration. These findings may have implications for radiation therapy in cancer treatment. Furthermore, given the presence of caspases throughout metazoa, the results could imply that preventing unwanted cell migration constitutes an ancient non-apoptotic function of these proteases (Gorelick-Ashkenazi, 2018).

    Xrp1 is a transcription factor required for cell competition-driven elimination of loser cells

    The elimination of unfit cells from a tissue is a process known in Drosophila and mammals as cell competition. In a well-studied paradigm 'loser' cells that are heterozygous mutant for a haploinsufficient ribosomal protein gene are eliminated from developing tissues via apoptosis when surrounded by fitter wild-type cells, referred to as 'winner' cells. However, the mechanisms underlying the induction of this phenomenon are not fully understood. This paper reports that a CCAAT-Enhancer-Binding Protein (C/EBP), Xrp1, which is known to help maintaining genomic stability after genotoxic stress, is necessary for the elimination of loser clones in cell competition. In loser cells, Xrp1 is transcriptionally upregulated by an autoregulatory loop and is able to trigger apoptosis -- driving cell elimination. Xrp1 acts in the nucleus to regulate the transcription of several genes that have been previously involved in cell competition. It is therefore speculated that Xrp1 might play a fundamental role as a molecular caretaker of the genomic integrity of tissues (Baillon, 2018).

    Tissues are composed by genetically heterogeneous cells as a result of the accumulation of different mutations over time. Unfit and potentially detrimental cells are eliminated from tissues via apoptosis triggered by a process known in both insects and mammals as cell competition. The eliminated cells, referred to as 'loser' cells, are normally viable and capable of growing, but are eliminated when surrounded by fitter, 'winner' cells. In Drosophila melanogaster, the majority of ribosomal protein genes (RPGs) are haploinsufficient (hRPGs). When one copy of an hRPG is removed, this gives rise to the 'Minute' phenotype characterized by a general developmental delay and improper bristle development. When intermingled with wild-type winner cells, cells heterozygous for an hRPG become losers and are eliminated via apoptosis. Various genetic manipulations of a tissue can result in different and widely documented cell competition responses. Several pathways, such as the BMP, Toll, Wnt, JAK/STAT, Ras/MAPK and Hippo pathways, have been implicated in cell competition, suggesting the existence of a complex framework of actions that serve to induce apoptosis and eliminate loser cells. However, what actually triggers elimination yet remains elusive. Multicellular organisms maintain genomic stability via the activation of DNA repair mechanisms to identify and correct DNA damages. During this process, cell cycle progression is arrested to prevent the expansion of damaged cells. However, when DNA repair fails, apoptosis is induced to eliminate irremediably damaged cells. The p53 transcription factor plays an evolutionarily conserved role in the induction of apoptosis following DNA damage, however evidence points towards the existence of alternative routes for the induction of apoptosis in response to DNA damage (Baillon, 2018).

    This study shows that, in a cell competition context, a possible alternative route to P53 for the induction of apoptosis goes via Xrp1, a gene encoding a b-ZIP DNA binding protein. The expression of Xrp1 is induced in various stress conditions, for instance in response to irradiation. Notably, Xrp1 mutant animals have been reported to have higher levels of loss-of-heterozygosity after ionizing radiation. Additionally Xrp1 plays a role in repair of DNA breaks after transposase cleavage. Therefore Xrp1 may have a role in sensing and responding to DNA damage (Baillon, 2018).

    This study report the discovery, in an EMS-based screen, of Xrp1 mutations that suppress the elimination of loser cells. This is consistent with earlier reports that proposed Xrp1 might affect cell competition. For the first time this study discerned how Xrp1 might regulate cell competition. Xrp1 is homologous to mammalian C/EBPs, a class of transcription factors that is known to autoregulate their own transcription, to prevent proliferation and induce apoptosis. It was further shown that Xrp1 expression is upregulated in loser cells in response to the removal of one copy of a haploinsufficient ribosomal protein gene, where, similarly to C/EBP homologs, it regulates its own expression via a positive autoregulatory loop, the expression of pro-apoptotic genes and that of other genes that were previously implicated in cell competition (Baillon, 2018).

    In order to identify genes whose function is necessary for the elimination of RPG heterozygous mutant loser cells, a forward genetic screen was performed using ethyl methanesulfonate (EMS) in Drosophila melanogaster. A mosaic system was designated that allows direct screening through the larval cuticle for the persistence of otherwise eliminated RpL19+/- loser clones. This enabled screening of a high number of animals for mutations that either dominantly (anywhere in the genome) or recessively (on the right arm of the third chromosome) suppress cell competition. The induction of a single somatic recombination event between two FLP recognition targets (FRTs) generates a RPG heterozygous mutant cell that becomes homozygous for the mutagenized right arm of the third chromosome. Loser clones are induced at the beginning of larval development (L1). If no suppressive mutation is present, clones are efficiently eliminated over time and thus undetectable by the end of the third instar larval stage (L3) when the screening is performed. 20,000 mutagenized genomes were screened for the presence of mutations that prevent the elimination of loser clones. Eleven heritable suppressors were obtained, and attention was focused on three of the strongest suppressors that did not display any obvious growth-related phenotype. Representative living larvae were analyzed for the presence of RpL19+/- GFP clones in the wing discs. RpL19+/- clones are eliminated and little or no signal is observed. Their elimination, however, is prevented when cells are not heterozygous mutant for RpL19 or when different Xrp1 mutations are additionally present. In the latter cases GFP signal is observed in wing discs (Baillon, 2018).

    Xrp1 suppressors did not belong to a lethal complementation group and the causative mutations were identified using a combination of positional mapping and whole-genome re-sequencing. In particular, three independent mutations in the introns of CG17836/Xrp1 were identified, all caused by substitutions of single nucleotides. These nucleotides are conserved within the Drosophila genus and inspection of the alignment revealed an embedment of these nucleotides in conserved sequence motifs. Of particular interest are the polypyrimidine motifs containing the nucleotide mutations in Xrp120 and Xrp108. These motifs flank the alternative first exon and are potential splice regulators. The CTCTCT motif in proximity of the 5' splice site of Xrp1 has been identified as a putative intronic splicing enhancer (ISE) predicted to serve as binding site for the polypyrimidine-tract binding protein (PTB) splicing regulator. The presence of these motifs prompted an investigation ito the consequences of the Xrp108 allele on exonic junctions. The most prominent effect of this allele is a strong and consistent reduction in the expression of two similar Xrp1 transcripts, RC and RE, which only differ in the composition of their 5' UTRs. They share the transcriptional start site and contain the same long open reading frame that codes for the short isoform of Xrp1 (Baillon, 2018).

    Then the behavior of RpL19+/- clones was checked in the presence and absence of Xrp1 function. To this end the twin spot MARCM system, which enables different marking twin clones generated by the same recombination event, was used. In the genetic set up, mCherry expression marks loser clones whereas two copies of GFP mark wild-type twin clones. As expected, RpL19+/- loser clones are eliminated from the tissue. Elimination is also observed when RpL19+/- cells within these clones are additionally mutant for Xrp108 but contain a transgene comprising the genomic region of Xrp1. Importantly, when Xrp1 mutations are not rescued cell competition-driven elimination of RpL19+/- losers no longer occurs. In particular, it was shown that the Xrp108 intronic mutation retrieved from the EMS screen is able to prevent loser cell elimination and that a similar result is obtained with a newly generated complete loss-of-function allele, Xrp161, as well as with Xrp126. Xrp161 contains a frame shift mutation upstream of the Xrp1 basic region-leucine zipper domain (b-ZIP), and is considered a null allele. Like other Xrp1 alleles analyzed it is homozygous viable and does not impair the development of mutant animals. To confirm that Xrp1 function is of general importance for the elimination of hRPG+/- cells, and not limited to RpL19+/- loser cells, the effect was tested of Xrp1 mutations on RpL14+/- loser clones (Fig. S2). Similarly to RpL19+/- cells, RpL14+/- cells are normally eliminated from wing discs during larval development. No elimination occurs if these cells express RpL14 from a transgene, or when Xrp1 is mutated (Xrp161) (Baillon, 2018).

    Since Xrp1 is transcriptionally induced in response to various forms of stress and since Xrp1 has been found to be upregulated in RpS3+/- wing discs when compared to WT discs, it was hypothesized that its expression is induced in loser clones as a result of the loss of a haploinsufficient ribosomal protein gene. A transcriptional reporter for Xrp1 was used (Xrp102515, containing a lacZ P-element) and it was found that Xrp1 expression is indeed upregulated in RpL19+/- cells, indicating that the upregulation of Xrp1 might play a crucial early role in the elimination of loser cells. In line with the recent report by Lee (2018), it was found that Xrp1 is upregulated in wing discs that are lacking one copy of a ribosomal protein gene, indicating that Xrp1's role in cell competition does not depend on clonality. In order to gain insights into this function the expression of Xrp1 was conditionally forced in the posterior half of the wing discs, and a massive induction of apoptosis was observed, as revealed by anti-cleaved caspase 3 staining (Baillon, 2018).

    To further explore this notion attempts were made to identify direct genomic targets of Xrp1 by chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) on wing imaginal discs. In order to do this, Xrp1 expression was induced in wing discs. The top targets revealed by ChIP-seq comprise a number of genes that are already implicated in cell competition, cell cycle regulation and apoptosis. Figure 4A shows a list of the most interesting genes that are bound by Xrp1. Among these were identified Xrp1 itself, suggesting the existence of a potential autoregulatory loop. To test this notion Xrp1 was overexpressed in the posterior compartment of the wing disc and the transcriptional behavior of Xrp1 was checked with the aforementioned Xrp1-lacZ reporter. The upregulation of lacZ expression was observed in response to Xrp1 overexpression, indicating that Xrp1 can boosts its own expression in a positive autoregulatory loop. These observations were checked by measuring mRNA levels of Xrp1 upon forced Xrp1 expression. With a similar strategy the response of other putative transcriptional targets from the ChIP-seq experiment were also checked. It was show that Xrp1 promotes the transcription of Dif, a Drosophila NFκB homolog gene that has previously been implicated in the cell competition-dependent induction of apoptosis via the induction of rpr transcription. puc, Upd3, Nedd4 and rad50 were also checked: all of these genes were upregulated upon induction of Xrp1 expression. puc, Upd3 and Nedd4 are involved in the JAK/STAT and Hippo signaling pathways, both of which have previously been implicated in cell competition. Rad50 is instead required for double strand break repair (Baillon, 2018).

    The most prominent sequence motif of Xrp1 derived from ChIP-seq data shows a strong similarity with the b-ZIP binding motif of the human C/EBP protein family. It was therefore checked whether Xrp1 shows homology to C/EBP transcription factors, being itself a bona fide transcription factor. Xrp1 was found to share a 40% identity with the human C/EBPs (PSI-BLAST). Phylogenetic reconstruction allowed us to recognize three Drosophila C/EBP homologs, one of which is Xrp1. Interestingly, human C/EBP-alpha is retained in the nucleolus and binds to ribosomal DNA34, a feature that may be evolutionarily conserved since Xrp1 binds rDNA loci with high affinity. The encoded rRNA is found in the nucleoli (Baillon, 2018).

    A working model is proposed in which Xrp1, under normal conditions, sits on rDNA in the nucleolus. In the presence of genotoxic stress or of a ribosomal imbalance, as in the context of Minute cell competition, Xrp1 acts nuclearly as a C/EBP transcription factor that stimulates its own transcription and the expression of pro-apoptotic target genes. When intermingled with wild-type cells, cells with only one copy of an hRPG are eliminated in a Xrp1-dependent manner. The deletion of one copy of the RpL19 gene is catalyzed by the Flp/FRT recombination system, which leaves no apparent lesion in the DNA. Therefore, the initial recruitment of Xrp1 into the nucleus may not depend on DNA damage per se, but rather on the unbalanced physiology of the cell resulting from the loss of one copy of the hRPG. The nucleolus is the site of ribosome biogenesis and a major stress sensor organelle. RpL19+/- cells experience a related nucleolar stress, since their nucleoli are enlarged as revealed by anti-fibrillarin staining. The most likely explanation for this is partially stalled ribosome assembly. Since genotoxic stress triggers Xrp1 expression, It is speculated that Xrp1 acts as a caretaker of genomic integrity. In support of this hypothesis, the growth of salvador-/- mutant tumor clones is suppressed by the concurrent loss of one copy of the RpL19 gene. However, this suppression fails in the absence of Xrp1 function, indicating that the presumptive protective function that RPGs haploinsufficiency provides can also operate within tumorous cells. In addition, according to a Monte-Carlo simulation, the likelihood that one hRPG locus becomes heterozygous mutant before any other gene gets mutated to homozygosity is very high. Together with the observation that hRPGs are broadly distributed within the genome, this further supports the potential role of Xrp1 as a caretaker of genomic integrity. Although further research is required to better elucidate this phenomenon, it is nevertheless proposed that RPG haploinsufficiency provides a simple, yet effective, mechanism to protect the organism from the emergence of potentially deleterious cells (Baillon, 2018).

    Culling less fit neurons protects against Amyloid-beta-induced brain damage and cognitive and motor decline

    Alzheimer's disease (AD) is the most common form of dementia, impairing cognitive and motor functions. One of the pathological hallmarks of AD is neuronal loss, which is not reflected in mouse models of AD. Therefore, the role of neuronal death is still uncertain. This study used a Drosophila AD model expressing a secreted form of human amyloid-beta42 peptide and showed that it recapitulates key aspects of AD pathology, including neuronal death and impaired long-term memory. Neuronal apoptosis is mediated by cell fitness-driven neuronal culling, which selectively eliminates impaired neurons from brain circuits. Removal of less fit neurons delays beta-amyloid-induced brain damage and protects against cognitive and motor decline, suggesting that contrary to common knowledge, neuronal death may have a beneficial effect in AD (Coelho, 2018).

    This study reports that expression of misfolding-prone toxic peptides linked to AD and Huntington's disease affects neuronal fitness and triggers competition between neurons, leading to increased activation of the FlowerLoseB isoform and Azot in Drosophila neural tissues. The results demonstrate that fitness fingerprints are important physiological mediators of neuronal death occurring in the course of neurodegenerative diseases (Coelho, 2018).

    This mechanism is associated with specific toxic peptides or with particular stages of the neurodegenerative disease, because competition is not elicited by expression of Parkinson-related α-Synuclein, for instance. The results suggest that the toxic effects of a given peptide correlate directly with the level of neuronal competition and death it induces (Coelho, 2018).

    Surprisingly, neuronal death was found to have a beneficial effect against β-amyloid-dependent cognitive and motor decline. This finding challenges the commonly accepted idea that neuronal death is detrimental at all stages of the disease progression. Most amyloid-induced neuronal apoptosis is beneficial and likely acts to remove damaged and/ or dysfunctional neurons in an attempt to protect neural circuits from aberrant neuronal activation and impaired synaptic transmission (Coelho, 2018).

    One curious observation in this study is that Ab42 induces cell death both autonomously and non-autonomously in clones of the eye disc. Dying cells co-localize with FlowerLoseB reporter both inside and outside of GFP-marked clones of the larva. It was observed that Ab42 peptide is secreted to regions outside of clone borders and accumulates at the basal side of the eye disc. The neurons of the eye disc that project their axons into the optic stalk through the basal side of the disc are likely affected by the accumulation of the toxic peptide, explaining the induction of cell death outside of clones (Coelho, 2018).

    Blocking apoptosis in Ab42 expressing flies by either azot silencing or overexpression of dIAP1 increases the number of vacuoles in the brains of these flies. This seems to be a counterintuitive observation, because one would expect that a reduction in apoptosis would result in fewer cells being lost and a reduction of neurodegenerative vacuoles. However, this observation can be conciliated with the current model: it is suspected that less fit neurons have impaired dendritic growth and inhibit the expansion of neighboring neurons. This inhibition would disappear once the unfit neuron is culled, allowing compensatory dendritic growth and neuropil extension (Coelho, 2018).

    Introduction of a single extra copy of azot was sufficient to prevent Ab42-induced motor and cognitive decline, which may suggest new venues for AD treatment that aim to support elimination of dysfunctional neurons at early stages of AD pathology. For example, in patients at early symptomatic stages, when cognitive impairment is first detected, enhancing physiological apoptotic pathways using Bcl-2 or Bcl-xL inhibitors, or promoting the cell competition pathway described in this study, may have strikingly beneficial effects (Coelho, 2018).

    Simu-dependent clearance of dying cells regulates macrophage function and inflammation resolution

    Macrophages encounter and clear apoptotic cells during normal development and homeostasis, including at numerous sites of pathology. Clearance of apoptotic cells has been intensively studied, but the effects of macrophage-apoptotic cell interactions on macrophage behaviour are poorly understood. Using Drosophila embryos, this study exploited the ease of manipulating cell death and apoptotic cell clearance in this model to identify that the loss of the apoptotic cell clearance receptor Six-microns-under (Simu) leads to perturbation of macrophage migration and inflammatory responses via pathological levels of apoptotic cells. Removal of apoptosis ameliorates these phenotypes, while acute induction of apoptosis phenocopies these defects and reveals that phagocytosis of apoptotic cells is not necessary for their anti-inflammatory action. Furthermore, Simu is necessary for clearance of necrotic debris and retention of macrophages at wounds. Thus, Simu is a general detector of damaged self and represents a novel molecular player regulating macrophages during resolution of inflammation (Roddie, 2019).

    Live cell tracking of macrophage efferocytosis during Drosophila embryo development in vivo

    Apoptosis of cells and their subsequent removal through efferocytosis occurs in nearly all tissues during development, homeostasis, and disease. However, it has been difficult to track cell death and subsequent corpse removal in vivo. This study developed a genetically encoded fluorescent reporter, CharON (Caspase and pH Activated Reporter, Fluorescence ON), that could track emerging apoptotic cells and their efferocytic clearance by phagocytes. Using Drosophila expressing CharON, multiple qualitative and quantitative features were uncovered of coordinated clearance of apoptotic corpses during embryonic development. When confronted with high rates of emerging apoptotic corpses, the macrophages displayed heterogeneity in engulfment behaviors, leading to some efferocytic macrophages carrying high corpse burden. Overburdened macrophages were compromised in clearing wound debris. These findings reveal known and unexpected features of apoptosis and macrophage efferocytosis in vivo (Raymond, 2022).

    Proto-pyroptosis: An ancestral origin for mammalian inflammatory cell death mechanism in Drosophila melanogaster

    Pyroptosis has been described in mammalian systems to be a form of programmed cell death that is important in immune function through the subsequent release of cytokines and immune effectors upon cell bursting. This form of cell death has been increasingly well-characterized in mammals and can occur using alternative routes however, across phyla, there has been little evidence for the existence of pyroptosis. This study provide evidence for an ancient origin of pyroptosis in an in vivo immune scenario in Drosophila melanogaster. Crystal cells, a type of insect blood cell, were recruited to wounds and ruptured subsequently releasing their cytosolic content in a caspase-dependent manner. This inflammatory-based programmed cell death mechanism fits the features of pyroptosis, never before described in an in vivo immune scenario in insects and relies on ancient apoptotic machinery to induce proto-pyroptosis. Further, this study unveiled key players upstream in the activation of cell death in these cells including the apoptosome which may play an alternative role akin to the inflammasome in proto-pyroptosis. Thus, Drosophila may be a suitable model for studying the functional significance of pyroptosis in the innate immune system (Dziedziech, 2021).

    Evidence for a novel function of Awd in maintenance of genomic stability

    The abnormal wing discs (awd) gene encodes the Drosophila homolog of NME1/NME2 metastasis suppressor genes. Awd acts in multiple tissues where its function is critical in establishing and maintaining epithelial integrity. This study analysed awd gene function in Drosophila epithelial cells using transgene-mediated RNA interference and genetic mosaic analysis. awd knockdown in larval wing disc epithelium leads to chromosomal instability (CIN) and induces apoptosis mediated by activation of c-Jun N-terminal kinase. Forced maintenance of Awd depleted cells, by expressing the cell death inhibitor p35, downregulates atypical protein kinase C and DE-Cadherin. Consistent with their loss of cell polarity and enhanced level of matrix metalloproteinase 1, cells delaminate from wing disc epithelium. Furthermore, the DNA content profile of these cells indicates that they are aneuploid. Overall, these data demonstrate a novel function for awd in maintenance of genomic stability. These results are consistent with other studies reporting that NME1 down-regulation induces CIN in human cell lines and suggest that Drosophila model could be successfully used to study in vivo the impact of NME/Awd induced genomic instability on tumour development and metastasis formation (Romani, 2017).

    Genomic stability is critical for cell survival and development and several cellular mechanisms act to maintain genomic integrity. Failure of these mechanisms underlies aging and can lead to malignancies such as cancer and age-related neurodegenerative diseases. Chromosomal instability (CIN) is a form of genomic instability that often leads to aneuploidy, a deleterious condition characterised by copy number changes affecting part or whole chromosomes. Several dysfunctions could lead to CIN. Defective activity of the spindle assembly checkpoint (SAC), a signalling pathway that blocks anaphase onset in response to mis-attachment of chromosomes to the mitotic spindle, leads to CIN and aneuploidy. Work in Drosophila showed that loss of function of SAC genes as well as loss of function of genes involved in spindle assembly, chromatin condensation and cytokinesis induce CIN. More recent work in larval disc epithelia has shown that down-regulation of these genes causes apoptotic cell death trough activation of the c-Jun N-terminal kinase (JNK) pathway. Interestingly, blocking CIN-induced apoptotic cell death induces tumourigenic behaviour including basement membrane degradation, cell delamination, tissue overgrowth and aneuploidy (Romani, 2017).

    The abnormal wing discs (awd) gene encodes the Drosophila homolog of NME1/2 metastasis suppressor genes. Awd is a well-known endocytic mediator whose function is required in multiple tissues during development. Genetic studies showed that Awd endocytic function ensures appropriate internalisation of chemotactic signalling receptors such as platelet-derived growth factor/VEGF receptor (PVR) and fibroblast growth factor receptor (FGFR) and thus it regulates invasion and cellular motility. Furthermore, this endocytic function regulates Notch receptor trafficking and is required for maintenance of epithelial integrity as it controls the turnover of adherens junction components in ovarian somatic follicle cells. Consistent with the high degree of functional conservation between Awd and its mammalian counterparts, recent studies have shown a role for the NME1/2 proteins in vesicular transport (Romani, 2017).

    This work has extended an analysis of the functional conservation between Awd and NME1/2 proteins. Since loss of NME1 gene function in human cell culture leads to polyploidy, this study have explored the role of Awd in maintenance of genomic stability. The data show that knockdown of awd in wing disc cells leads to CIN and to the CIN-induced biological responses mediated through JNK activation. Furthermore, when combined with block of apoptosis, down-regulation of awd leads to cell delamination and aneuploidy. Thus, the results of this in vivo analysis show a novel function for awd in maintenance of genomic stability (Romani, 2017).

    Depletion of Awd triggers JNK-mediated cell death of wing disc cells and blocking the cell death machinery results in aneuploidy and cell delamination without overt hyperproliferative effect. Overgrowth of wing disc hosting aneuploid cells is due to activation of the JNK pathway that promotes expression of Wingless (Wg) upon blocking of apoptotic cell death. Wg is a mitogenic molecule required in the imaginal discs for growth and patterning and its expression in the aneuploid, delaminating CIN cells triggers growth of neighbouring non-delaminating cells. However, awd J2A4 mutant wing disc cells do not express Wg as a consequence of faulty Notch signalling; therefore, these cells cannot promote hyperplasia of the surrounding tissue. Furthermore, lack of hyperproliferation is also observed when an aneuploid condition arises from impaired activity of genes controlling karyokinesis. The diaphanous gene (dia) codes for an actin-regulatory molecule that is required during acto-myosin driven contraction of metaphase furrows. Simultaneous depletion of dia gene expression and blocking of apoptosis do not lead to hyperplastic growth probably due to defective karyokinesis. Intriguingly, Awd is a microtubule-associated nucleoside diphosphate kinase that converts GDP to GTP and the analysis of awd mutant larval brain showed mitotic defects correlated with defective microtubule polymerisation. This raises the possibility that the Awd kinase function plays a role in GTP supply to proteins such as Orbit that are required for stabilisation of spindle microtubules (Romani, 2017).

    Two lines of evidence further support the hypothesis that Awd could be involved in karyokinesis. The first comes from studies showing that endosome trafficking and transport to the intercellular bridges of dividing cells plays a critical role during abscission, the last step of karyokinesis. In addition, remodelling the of plasma membrane that underlies nuclear divisions in the syncytial embryo and cellularisation also requires endocytosis. Embryonic cellularisation requires the dynamin encoded by the shibire (shi) locus and Rab5 GTPase function, since loss of function of either genes arrests ingression of metaphase furrows. Awd functionally interacts with shi locus and Awd is also required for Rab5 function in early endosomes. Thus, a possible role for Awd in cytokinesis should be considered (Romani, 2017).

    The second line of evidence comes from studies on NME1, the human homolog of awd gene. This metastasis suppressor gene shares about 78% of amino acid identity with the awd gene. Down-regulation of NME1 gene expression in diploid cells results in cytokinesis failure and leads to tetraploidy. The in vivo results show that Awd plays a role in maintenance of genomic stability, confirming the high degree of conservation between NME1 and Awd proteins. Drosophila studies have already been crucial in identification of NME1 function in epithelial morphogenesis, and the present work shows that this function can be a useful model for impact on tumour development and progression (Romani, 2017).

    Decoupling developmental apoptosis and neuroblast proliferation in Drosophila

    Cell proliferation and cell death are opposing but fundamental aspects of development that must be tightly controlled to ensure proper tissue organization and organismal health. Developmental apoptosis of abdominal neuroblasts in the Drosophila ventral nerve cord is controlled by multiple upstream spatial and temporal signals, which have also been implicated in control of cell proliferation. It has therefore remained unclear whether developmental apoptosis is linked to active cell proliferation. Previous investigations into this topic have focused on the effect of cell cycle arrests on exogenous induction of apoptosis, and thus have not addressed whether potential effects of the cell cycle lie with the sensing of damage signals or the execution of apoptosis itself. This report shows that developmental apoptosis is not inhibited by cell cycle arrest, and that endogenous cell death occurs independently of cell cycle phase. Ectopic neuroblasts rescued from cell death retain the competency to respond to quiescence cues at the end of embryogenesis. In addition, multiple quiescence types were observed in neuroblasts, and cell death mutant embryos display a specific loss of presumptive G2 quiescent abdominal neuroblasts at the end of embryogenesis. This study demonstrates that upstream control of neuroblast proliferation and apoptosis represent independent mechanisms of regulating stem cell fate, and that execution of apoptosis occurs in a cell cycle-independent manner. These findings also indicate that a subset of G2Q-fated abdominal neuroblasts are eliminated from the embryo through a non-apoptotic mechanism (Harding, 2019).

    Drosophila FGFR/Htl signaling shapes embryonic glia to phagocytose apoptotic neurons

    Glial phagocytosis of apoptotic neurons is crucial for development and proper function of the central nervous system. Relying on transmembrane receptors located on their protrusions, phagocytic glia recognize and engulf apoptotic debris. Like vertebrate microglia, Drosophila phagocytic glial cells form an elaborate network in the developing brain to reach and remove apoptotic neurons. However, the mechanisms controlling creation of the branched morphology of these glial cells critical for their phagocytic ability remain unknown. This study demonstrated that during early embryogenesis, the Drosophila fibroblast growth factor receptor (FGFR) Heartless (Htl) and its ligand Pyramus are essential in glial cells for the formation of glial extensions, the presence of which strongly affects glial phagocytosis of apoptotic neurons during later stages of embryonic development. Reduction in Htl pathway activity results in shorter lengths and lower complexity of glial branches, thereby disrupting the glial network. This work thus illuminates the important role Htl signaling plays in glial subcellular morphogenesis and in establishing glial phagocytic ability (Ayoub, 2023).

    The dual role of heme oxygenase in regulating apoptosis in the nervous system of Drosophila melanogaster

    Accumulating evidence from mammalian studies suggests the dual-faced character of heme oxygenase (HO) in oxidative stress-dependent neurodegeneration. The present study aimed to investigate both neuroprotective and neurotoxic effects of heme oxygenase after the Ho gene chronic overexpression or silencing in neurons of Drosophila melanogaster. The results showed early deaths and behavioral defects after pan-neuronal Ho overexpression, while survival and climbing in a strain with pan-neuronal Ho silencing were similar over time with its parental controls. It was also found that Ho can be pro-apoptotic or anti-apoptotic under different conditions. In young (7-day-old) flies, both the cell death activator gene (hid) expression and the initiator caspase Dronc activity increased in heads of flies when ho expression was changed. In addition, various expression levels of ho produced cell-specific degeneration. Dopaminergic (DA) neurons and retina photoreceptors are particularly vulnerable to changes in Ho expression. In older (30-day-old) flies, no further increase was detected in hid expression or enhanced degeneration, however, high activity of the initiator caspase was still observed. In addition, curcumin, a biologically active polyphenolic compound found in turmeric, was used to further show the involvement of neuronal Ho in the regulation of apoptosis. Under normal conditions, curcumin induced both the expression of Ho and hid, which was reversed after exposure to high-temperature stress and when supplemented in flies with Ho silencing. These results indicate that neuronal Ho regulates apoptosis and this process depends on Ho expression level, age of flies, and cell type (Abaquita, 2023).

    Long-term sevoflurane exposure resulted in temporary rather than lasting cognitive impairment in Drosophila

    Sevoflurane is the primary inhaled anesthetic used in pediatric surgery. It has been the focus of research since animal models studies found that it was neurotoxic to the developing brain two decades ago. However, whether pediatric general anesthesia can lead to permanent cognitive deficits remained a subject of heated debate. Therefore, this study aims to determine the lifetime neurotoxicity of early long-time sevoflurane exposure using a short-life-cycle animal model, Drosophila melanogaster. To investigate this question, the lifetime changes of two-day-old flies' learning and memory abilities after anesthesia with 3 % sevoflurane for 6 h by the T-maze memory assay. Apoptosis, levels of ATP and ROS, and related genes were evaluated in the fly head. The results suggest that 6 h 3 % sevoflurane exposure at a young age can only induce transient neuroapoptosis and cognitive deficits around the first week after anesthesia. But this brain damage recedes with time and vanishes in late life. It was also found that the mRNA level of caspases and Bcl-2, ROS level, and ATP level increased during this temporary neuroapoptosis process. And mRNA levels of antioxidants, such as SOD2 and CAT, increased and decreased simultaneously with the rise and fall of the ROS level, indicating a possible contribution to the recovery from the sevoflurane impairment. In conclusion, these results suggest that one early prolonged sevoflurane-based general anesthesia can induce neuroapoptosis and learning and memory deficit transiently but not permanently in Drosophila (Liu, 2023).

    Ensheathing glia promote increased lifespan and healthy brain aging

    Glia have an emergent role in brain aging and disease. In the Drosophila melanogaster brain, ensheathing glia function as phagocytic cells and respond to acute neuronal damage, analogous to mammalian microglia. Changes in glia composition over the life of ants and fruit flies have been reported, including a decline in the relative proportion of ensheathing glia with time. How these changes influence brain health and life expectancy is unknown. This study shows that ensheathing glia but not astrocytes decrease in number during Drosophila melanogaster brain aging. The remaining ensheathing glia display dysregulated expression of genes involved in lipid metabolism and apoptosis, which may lead to lipid droplet accumulation, cellular dysfunction, and death. Inhibition of apoptosis rescued the decline of ensheathing glia with age, improved the neuromotor performance of aged flies, and extended lifespan. Furthermore, an expanded ensheathing glia population prevented amyloid-beta accumulation in a fly model of Alzheimer's disease and delayed the premature death of the diseased animals. These findings suggest that ensheathing glia play a vital role in regulating brain health and animal longevity (Sheng, 2023).

    Non-muscle MYH10/myosin IIB recruits ESCRT-III to participate in autophagosome closure to maintain neuronal homeostasis

    Dysfunction of the endosomal sorting complex required for transport (ESCRT) has been linked to frontotemporal dementia (FTD) due in part to the accumulation of unsealed autophagosomes. However, the mechanisms of ESCRT-mediated membrane closure events on phagophores remain largely unknown. This study found that partial knockdown of non-muscle MYH10/myosin IIB/zip rescues neurodegeneration in both Drosophila and human iPSC-derived cortical neurons expressing FTD-associated mutant CHMP2B, a subunit of ESCRT-III. It was also found that MYH10 binds and recruits several autophagy receptor proteins during autophagosome formation induced by mutant CHMP2B or nutrient starvation. Moreover, MYH10 interacted with ESCRT-III to regulate phagophore closure by recruiting ESCRT-III to damaged mitochondria during PRKN/parkin-mediated mitophagy. Evidently, MYH10 is involved in the initiation of induced but not basal autophagy and also links ESCRT-III to mitophagosome sealing, revealing novel roles of MYH10 in the autophagy pathway and in ESCRT-related FTD pathogenesis (Jun, 2023).

    Potent New Targets for Autophagy Enhancement to Delay Neuronal Ageing

    Autophagy is a lysosomal-dependent degradation process of eukaryotic cells responsible for breaking down unnecessary and damaged intracellular components. This study aimed to uncover new regulatory points where autophagy could be specifically activated and tested these potential drug targets in neurodegenerative disease models of Drosophila melanogaster. One possible way to activate autophagy is by enhancing autophagosome-lysosome fusion that creates the autolysosome in which the enzymatic degradation happens. The HOPS (homotypic fusion and protein sorting) and SNARE (Snap receptor) protein complexes regulate the fusion process. The HOPS complex forms a bridge between the lysosome and autophagosome with the assistance of small GTPase proteins. Thus, small GTPases are essential for autolysosome maturation, and among these proteins, Rab2 (Ras-associated binding 2), Rab7, and Arl8 (Arf-like 8) are required to degrade the autophagic cargo. For these experiments, Drosophila melanogaster was used as a model organism. Nerve-specific small GTPases were silenced and overexpressed. The effects were examined of these genetic interventions on lifespan, climbing ability, and autophagy. Finally, the activation of small GTPases was also studied in a Parkinson's disease model. The results revealed that GTP-locked, constitutively active Rab2 (Rab2-CA) and Arl8 (Arl8-CA) expression reduces the levels of the autophagic substrate p62/Ref(2)P in neurons, extends lifespan, and improves the climbing ability of animals during ageing. However, Rab7-CA expression dramatically shortens lifespan and inhibits autophagy. Rab2-CA expression also increases lifespan in a Parkinson's disease model fly strain overexpressing human mutant (A53T) α-synuclein protein. Data provided by this study suggests that Rab2 and Arl8 serve as potential targets for autophagy enhancement in the Drosophila nervous system (Szinyakovics, 2023).

    Atg2 Regulates Cellular and Humoral Immunity in Drosophila

    Autophagy is a process that promotes the lysosomal degradation of cytoplasmic proteins and is highly conserved in eukaryotic organisms. Autophagy maintains homeostasis in organisms and regulates multiple developmental processes, and autophagy disruption is related to human diseases. However, the functional roles of autophagy in mediating innate immune responses are largely unknown. This study sought to understand how Atg2, an autophagy-related gene, functions in the innate immunity of Drosophila melanogaster. The results showed that a large number of melanotic nodules were produced upon inhibition of Atg2. In addition, inhibiting Atg2 suppressed the phagocytosis of latex beads, Staphylococcus aureus and Escherichia coli; the proportion of Nimrod C1 (one of the phagocytosis receptors)-positive hemocytes also decreased. Moreover, inhibiting Atg2 altered actin cytoskeleton patterns, showing longer filopodia but with decreased numbers of filopodia. The expression of AMP-encoding genes was altered by inhibiting Atg2. Drosomycin was upregulated, and the transcript levels of Attacin-A, Diptericin and Metchnikowin were decreased. Finally, the above alterations caused by the inhibition of Atg2 prevented flies from resisting invading pathogens, showing that flies with low expression of Atg2 were highly susceptible to Staphylococcus aureus and Erwinia carotovora carotovora 15 infections. In conclusion, Atg2 regulated both cellular and humoral innate immunity in Drosophila. We have identified Atg2 as a crucial regulator in mediating the homeostasis of immunity, which further established the interactions between autophagy and innate immunity (Qin, 2023).

    Natural genetic variation screen in Drosophila identifies Wnt signaling, mitochondrial metabolism, and redox homeostasis genes as modifiers of apoptosis

    Apoptosis is the primary cause of degeneration in a number of neuronal, muscular, and metabolic disorders. These diseases are subject to a great deal of phenotypic heterogeneity in patient populations, primarily due to differences in genetic variation between individuals. This creates a barrier to effective diagnosis and treatment. Understanding how genetic variation influences apoptosis could lead to the development of new therapeutics and better personalized treatment approaches. This study examined the impact of the natural genetic variation in the Drosophila Genetic Reference Panel (DGRP) on two models of apoptosis-induced retinal degeneration: overexpression of p53 or reaper (rpr). A number of known apoptotic, neural, and developmental genes were identified as candidate modifiers of degeneration. Gene Set Enrichment Analysis (GSEA) was used to identify pathways that harbor genetic variation that impact these apoptosis models, including Wnt signaling, mitochondrial metabolism, and redox homeostasis. Finally, many of these candidates were demonstrated to have a functional effect on apoptosis and degeneration. These studies provide a number of avenues for modifying genes and pathways of apoptosis-related disease (Palu, 2019).

    Two-factor specification of apoptosis: TGF-beta signaling acts cooperatively with ecdysone signaling to induce cell- and stage-specific apoptosis of larval neurons during metamorphosis in Drosophila melanogaster

    Developmentally regulated programmed cell death (PCD) is one of the key cellular events for precise controlling of neuronal population during postembryonic development of the central nervous system. Previous work has shown that a group of corazonin-producing peptidergic neurons (vCrz) undergo apoptosis in response to ecdysone signaling via ecdysone receptor (EcR)-B isoforms and Ultraspiracle during early phase of metamorphosis. Further utilizing genetic, transgenic, and mosaic analyses, it was found that TGF-beta signaling mediated by a glia-produced ligand, Myoglianin, type-I receptor Baboon (particularly Babo-A isoform) and dSmad2, is also required autonomously for PCD of the vCrz neurons. These studies show that TGF-beta signaling is not acting epistatically to EcR or vice versa. It was also shown that ectopic expression of a constitutively active phosphomimetic form of dSmad2 (dSmad2(PM)) is capable of inducing premature death of vCrz neurons in larva but not other larval neurons. Intriguingly, the dSmad2(PM)-mediated killing is completely suppressed by coexpression of a dominant-negative form of EcR (EcR(DN)), suggesting that EcR function is required for the proapoptotic dSmad2(PM) function. Based on these data, it is suggested that TGF-beta and ecdysone signaling pathways act cooperatively to induce vCrz neuronal PCD. It is proposed that this type of two-factor authentication is a key developmental strategy to ensure the timely PCD of specific larval neurons during metamorphosis (Wang, 2019).

    Effects of cell death-induced proliferation on a cell competition system

    Cell death-induced proliferation (CDIP) is a phenomenon in which cell death activates neighboring cells and promotes their proliferation. It was first reported as "compensatory proliferation" in injured tissues, which functions to maintain normal tissues. On the other hand, this phenomenon also affects potentially tumorigenic mutant cells and promotes tumorigenesis. This discrepancy may complicate the understanding of a phenomenon called "cell competition" observed in a system consisting of wild-type (WT) cells and mutant cells in a single-layer tissue. In this system, the WT cells induce cell death in the adjacent mutant cells to eliminate them. Therefore, it is believed that CDIP serves WT cells by compensating the space previously occupied by mutant cells. On the other hand, CDIP may contribute to the expansion of a potentially tumorigenic mutant clone because this clone activates itself. With the aim to investigate the role of CDIP in cell competition, a mathematical model was constructed in this study by introducing a CDIP effect into the population-based cell competition model that was proposed in previous work. In contrast to the above-mentioned first expectation, the model suggests that the CDIP of WT cells that is derived from cell competition does not affect the fate whether it follows formation of normal tissue or overgrowth of a mutant clone after cell competition. It should be noted, however, that CDIP accelerates the speed of normal tissue formation; only this point is in agreement with expectations. In contrast, the CDIP of mutant cells that is derived from either autonomous cell death or cell competition helps mutant cells to survive (Nishikawa, 2019).

    NCBP2 modulates neurodevelopmental defects that are rescued by overexpression of the apoptosis inhibitors Diap1 and xiapof the 3q29 deletion in Drosophila and Xenopus laevis models

    The 1.6 Mbp deletion on chromosome 3q29 is associated with a range of neurodevelopmental disorders, including schizophrenia, autism, microcephaly, and intellectual disability. Despite its importance towards neurodevelopment, the role of individual genes, genetic interactions, and disrupted biological mechanisms underlying the deletion have not been thoroughly characterized. This study used quantitative methods to assay Drosophila melanogaster and Xenopus laevis models with tissue-specific individual and pairwise knockdown of 14 homologs of genes within the 3q29 region. Developmental, cellular, and neuronal phenotypes were identified for multiple homologs of 3q29 genes, potentially due to altered apoptosis and cell cycle mechanisms during development. Using the fly eye, screening was performed for 314 pairwise knockdowns of homologs of 3q29 genes and 44 interactions between pairs of homologs and 34 interactions with other neurodevelopmental genes were identified. Interestingly, NCBP2 homologs in Drosophila (Cbp20) and X. laevis (ncbp2) enhanced the phenotypes of homologs of the other 3q29 genes, leading to significant increases in apoptosis that disrupted cellular organization and brain morphology. These cellular and neuronal defects were rescued with overexpression of the apoptosis inhibitors Diap1 and xiap in both models, suggesting that apoptosis is one of several potential biological mechanisms disrupted by the deletion. NCBP2 was also highly connected to other 3q29 genes in a human brain-specific interaction network, providing support for the relevance of these results towards the human deletion. Overall, this study suggests that NCBP2-mediated genetic interactions within the 3q29 region disrupt apoptosis and cell cycle mechanisms during development (Singh, 2020).

    Xrn1/Pacman affects apoptosis and regulates expression of hid and reaper

    Programmed cell death, or apoptosis, is a highly conserved cellular process that is crucial for tissue homeostasis under normal development as well as environmental stress. Misregulation of apoptosis is linked to many developmental defects and diseases such as tumour formation, autoimmune diseases and neurological disorders. This paper shows a novel role for the exoribonuclease Pacman/Xrn1 in regulating apoptosis. Using Drosophila wing imaginal discs as a model system, a null mutation in pacman was demonstrated to result in small imaginal discs as well as lethality during pupation. Mutant wing discs show an increase in the number of cells undergoing apoptosis, especially in the wing pouch area. Compensatory proliferation also occurs in these mutant discs, but this is insufficient to compensate for the concurrent increase in apoptosis. The phenotypic effects of the pacman null mutation are rescued by a deletion that removes one copy of each of the pro-apoptotic genes reaper, hid and grim, demonstrating that pacman acts through this pathway. The null pacman mutation also results in a significant increase in the expression of the pro-apoptotic mRNAs, hid and reaper, with this increase mostly occurring at the post-transcriptional level, suggesting that Pacman normally targets these mRNAs for degradation. These results uncover a novel function for the conserved exoribonuclease Pacman and suggest that this exoribonuclease is important in the regulation of apoptosis in other organisms (Waldron, 2015).

    Apoptosis is a key process in developmental pathways and also in cancer. This study has generated a null mutation in pacman (pcm14 ) and used this to show that Pacman can control apoptosis in wing imaginal discs by regulating levels of hid and reaper mRNAs. Use of the Df(3L)H99 deletion, which removes one copy of the hid, grim and reaper genes, largely rescues the effect of the pcm14 mutation on growth of the wing imaginal discs and on developmental timing. However, the Df(3L)H99 deletion (Df(3L)H99/+) does not rescue the lethality of the pcm14 mutation. This suggests that there may be other targets of Pacman that are misregulated in pcm14 larvae or pupae (Waldron, 2015).

    Mutant wing discs are proportionately reduced in size, even though the majority of apoptosis occurs in the wing pouch. The data also show that Pacman is expressed over the entire disc and pcm14 /pcm14 mutant clones are smaller than their wild-type twin spots throughout the disc. It is possible that apoptosis is occurring throughout the disc in earlier stages of development but is restricted to the wing pouch during L3. The data is consistent with other studies reporting that cells within the wing pouch are particularly sensitive to apoptosis, perhaps due to expression of particular apoptotic regulators in that region. The co-ordinate growth of the wing disc, even though apoptosis is occurring in a particular region of the disc, is likely to be due to long range signalling via morphogens which control overall patterning and growth of the wing disc. For example, Decapentaplegic (Dpp), a bone morphogenetic protein (BMP) functions as a long range morphogen to control patterning and growth. Furthermore, the Aegerter-Wilmsen model which explains how growth is constant throughout the disc suggests that growth of the peripheral cells within the disc is caused by stretching of the cells as a result of growth at the centre of the disc (see Aegerter-Wilmsen, 2012). Therefore, reduced growth at the centre of the disc, caused by apoptosis specifically in the pouch, is likely to cause reduced growth of the whole disc (Waldron, 2015).

    The results also show that the pcm14 mutation induces cell proliferation as well as apoptosis. Apoptosis-induced compensatory proliferation is known to occur to maintain tissue homeostasis so that damaged tissues can be replaced allowing the organ to maintain its normal size. In Drosophila, this occurs via the initiator caspase Dronc which induces compensatory proliferation as well as apoptosis. Since Dronc is activated by Hid and Reaper, the increase in hid and reaper mRNA in pcm14 cells is consistent with increased activity of Dronc. Nevertheless, the 51%-54% increase in cell division in the pcm14 wing imaginal discs is insufficient to compensate for the concurrent increase in apoptosis because the wing discs fail to develop and differentiate, leading to death of the pupa. This failure of the wing discs to regenerate could be explained by there being prolonged apoptosis in the pcm14 wing imaginal discs, whereas other experiments have induced a pulse of apoptosis, allowing time for the wing disc to recover (Waldron, 2015).

    The above results are consistent with reaper and hid being translated from the upregulated reaper and hid transcripts in pcm14 mutants. This would imply that these transcripts are both capped and polyadenylated. Biochemical analyses have shown that the less structured C-terminal domain of Pacman/Xrn1 includes short sections of conserved amino acids which bind co-factors such as the decapping protein Dcp1. Dcp1 associates with the decapping enzyme Dcp2, therefore coupling decapping to 5'-3' degradation. In pcm14 cells where no Pacman is present, decapping would therefore be expected to be impaired, which is consistent with our data. The alternative and/or additional hypothesis is that reaper and hid are being translated in a cap independent manner. Indeed the 5' UTRs of these genes have been shown to contain functional Internal Ribosome Entry Sites (IRES) and are still able to undergo translation in cells in which cap dependent translation is impeded (Waldron, 2015).

    The above molecular mechanisms also are consistent with the 'dominant negative' effect seen when the nuclease-dead version of Pacman is expressed in a pcm14 mutant background. In Drosophila tissue culture cells, over-expression of catalytically inactive Pacman inhibited both decapping and degradation of a reporter RNA leading to an accumulation of capped fragments (Braun, 2012). Therefore the dominant negative effect could result from the sequestration of the Decapping protein Dcp1 together with lack of exonuclease activity. Expressing a 'nuclease dead' Pacman in pcm14 cells would not rescue any exoribonuclease activity but could impair decapping further. The results therefore support the model (Jones, 2012) that Pacman/Xrn1 normally assembles a complex of 5'-3' degradation factors including Dcp1 to provide a multicomponent complex which decaps and then degrades specific RNAs in a 5'-3' direction (Waldron, 2015).

    The data, using natural tissue rather than immortalised tissue culture cells, supports the idea that there is a network of RNA-protein interactions contributing to apoptosis and proliferation. This idea is supported by work on the deadenylases Ccr4a and Ccr4b which can affect cell survival in MCF7 human breast cancer cells. Further, the RNA-binding protein HuR (homologue of Elav in Drosophila) has recently been shown to be cleaved in HeLa cells during caspase-mediated apoptosis with the two cleavage fragments binding to and stabilising caspase 9 mRNA, thus promoting apoptosis. The current data showing that the exoribonuclease Pacman is also involved in the control of apoptosis suggests a key role for the 5'-3' degradation pathway in the regulation of apoptosis (Waldron, 2015).

    What are the mechanisms by which Pacman might be affecting the levels of mature hid and reaper mRNA? The simplest hypothesis is that Pacman is normally targeted to hid and reaper mRNA, resulting in degradation of these mRNAs. This targeting could be accomplished by specific RNA binding proteins and/or miRNAs binding to the 3' UTRs of hid and reaper mRNAs and directing them to the 5'-3' degradation machinery. The 3' untranslated regions of hid and reaper contain many predicted and validated miRNA binding sites for miRNAs. For example, the miRNA bantam has been shown to bind to the 3' UTR of hid mRNA, thus regulating its expression. In addition, miR-2 is known to bind to the 3' UTR of reaper, repressing its translation and directing it to P-body-like structures. A possible model to explain the results is that reaper and hid mRNAs are normally unstable because they are directed to the 5'-3' degradation complex by miRNAs binding to their 3' UTRs. In wild-type cells, these RNAs are rapidly decapped by decapping enzymes associated with Pacman and then degraded in a 5'-3' direction. In the Pacman mutant, these mRNAs are not efficiently degraded because of the absence of Pacman. It is also possible that reaper and hid are particularly affected by loss of Pacman because the presence of IRES sequences within their 5' UTRs means that these RNAs can be translated even if they are decapped. In a pacman mutant, these decapped RNAs may still be translated to produce Reaper and Hid protein. The exact mechanisms whereby Pacman regulates these mRNAs will require further research (Waldron, 2015).

    Functional integration of "undead" neurons in the olfactory system

    Programmed cell death (PCD) is widespread during neurodevelopment, eliminating the surpluses of neuronal production. Using the Drosophila olfactory system, this study examined the potential of cells fated to die to contribute to circuit evolution. Inhibition of PCD is sufficient to generate new cells that express neural markers and exhibit odor-evoked activity. These "undead" neurons express a subset of olfactory receptors that is enriched for relatively recent receptor duplicates and includes some normally found in different chemosensory organs and life stages. Moreover, undead neuron axons integrate into the olfactory circuitry in the brain, forming novel receptor/glomerular couplings. Comparison of homologous olfactory lineages across drosophilids reveals natural examples of fate change from death to a functional neuron. Last, evidence is provided that PCD contributes to evolutionary differences in carbon dioxide-sensing circuit formation in Drosophila and mosquitoes. These results reveal the remarkable potential of alterations in PCD patterning to evolve new neural pathways (Prieto-Godino, 2020)

    Three-tier regulation of cell number plasticity by neurotrophins and Tolls in Drosophila

    Cell number plasticity is coupled to circuitry in the nervous system, adjusting cell mass to functional requirements. In mammals, this is achieved by neurotrophin (NT) ligands, which promote cell survival via their Trk and p75NTR receptors and cell death via p75NTR and Sortilin. Drosophila NTs (DNTs; see NT1) bind Toll receptors (see Toll-6 & Toll-7) instead to promote neuronal survival, but whether they can also regulate cell death is unknown. This study show that DNTs and Tolls can switch from promoting cell survival to death in the central nervous system (CNS) via a three-tier mechanism. First, DNT cleavage patterns result in alternative signaling outcomes. Second, different Tolls can preferentially promote cell survival or death. Third, distinct adaptors downstream of Tolls can drive either apoptosis or cell survival. Toll-6 promotes cell survival via MyD88-NF-κB and cell death via Wek-Sarm-JNK. The distribution of adaptors changes in space and time and may segregate to distinct neural circuits. This novel mechanism for CNS cell plasticity may operate in wider contexts (Foldi, 2017).

    Balancing cell death and cell survival enables structural plasticity and homeostasis, regeneration, and repair and fails in cancer and neurodegeneration. In the nervous system, cell number plasticity is linked to neural circuit formation, adjusting neuronal number to functional requirements. In mammals, the neurotrophin (NT) protein family [NGF, brain-derived neurotrophic factor (BDNF), NT3, and NT4] regulates neuronal number through two mechanisms. First, full-length pro-NTs, comprised of a disordered prodomain and a cystine-knot (CK) domain, induce cell death; in contrast, mature NTs formed of CK dimers promote cell survival. Second, pro-NTs bind p75NTR and Sortilin receptors, inducing apoptosis via JNK signaling, whereas mature NTs bind p75NTR, promoting cell survival via NF-κB and TrkA, B, and C, promoting cell survival via PI3K/AKT and MAPK/ERK. As the NTs also regulate connectivity and synaptic transmission, they couple the regulation of cell number to neural circuitry and function, enabling structural brain plasticity. There is abundant evidence that cell number plasticity occurs in Drosophila melanogaster central nervous system (CNS) development, with neurotrophic factors including NTs and mesencephalic astrocyte-derived neurotrophic factor (MANF), but fruit flies lack p75NTR and Trk receptors, raising the question of how this is achieved in the fly. Finding this out is important, as it could lead to novel mechanisms of structural plasticity for both flies and humans (Foldi, 2017).

    The Drosophila NTs (DNTs) Spätzle (Spz), DNT1, and DNT2 share with mammalian NTs the characteristic structure of a prodomain and a conserved CK of 13-15 kD, which forms a disulfide-linked dimer. Spz resembles NGF biochemically and structurally, and the binding of its Toll-1 receptor resembles that of NGF to p75NTR. DNT1 (also known as spz2) was discovered by homology to BDNF, and DNT2 (also known as spz5) as a paralogue of spz and DNT1. DNT1 and 2 promote neuronal survival, and DNT1 and 2, Spz, and Spz3 are required for connectivity and synaptogenesis. Spz, DNT1, and DNT2 are ligands for Toll-1, -7, and -6, respectively, which function as NT receptors and promote neuronal survival, circuit connectivity, and structural synaptic plasticity. Tolls belong to the Toll receptor superfamily, which underlies innate immunity. There are nine Toll paralogues in flies, of which only Toll-1, -5, -7, and -9 are involved in immunity. Tolls are also involved in morphogenesis, cell competition, and epidermal repair. Whether DNTs and Tolls can balance cell number plasticity is unknown (Foldi, 2017).

    Like the p75NTR receptor, Toll-1 activates NF-κB (a potent neuronal prosurvival factor with evolutionarily conserved functions also in structural and synaptic plasticity) signaling downstream. Toll-1 signaling involves the downstream adaptor MyD88, which forms a complex with Tube and Pelle. Activation of Toll-1 triggers the degradation of the NF-κB inhibitor Cactus, enabling the nuclear translocation of the NF-κB homologues Dorsal and Dorsal-related immunity factor (Dif), which function as transcription factors. Other Tolls have also been suggested to activate NF-κB. However, only Toll-1 has been shown to bind MyD88, raising the question of how the other Tolls signal in flies (Foldi, 2017).

    Whether Tolls regulate cell death is also obscure. Toll-1 activates JNK, causing apoptosis, but its expression can also be activated by JNK to induce nonapoptotic cell death. Toll-2, -3, -8, and -9 can induce apoptosis via NF-κB and dSarm independently of MyD88 and JNK. However, in the CNS, dSarm induces axonal degeneration, but there is no evidence that it can promote apoptosis in flies. In other animals, Sarm orthologues are inhibitors of Toll signaling and MyD88, but there is no evidence that dSarm is an inhibitor of MyD88 in Drosophila. Thus, whether or how Tolls may regulate apoptosis in flies is unclear (Foldi, 2017).

    In the mammalian brain, Toll-like receptors (TLRs) are expressed in neurons, where they regulate neurogenesis, apoptosis, and neurite growth and collapse in the absence of any insult. However, their neuronal functions have been little explored, and their endogenous ligands in neurons remain unknown (Foldi, 2017).

    Because Toll-1 and p75NTR share common downstream signaling pathways and p75NTR can activate NF-κB to promote cell survival and JNK to promote cell death, this study asked whether the DNTs and their Toll receptors could have dual roles controlling cell survival and death in the Drosophila CNS (Foldi, 2017).

    In the first regulatory tier, each DNT has unique features conducive to distinctive functions. Spz, DNT1, and DNT2 share with the mammalian NTs the unequivocal structure of the CK domain unique to this protein family. However, DNT1, DNT2, and Spz have distinct prodomain features and are processed differently, leading to distinct cellular outcomes. Spz is only secreted full length and cleaved by serine proteases. DNT1 and 2 are cleaved intracellularly by conserved furins. In cell culture, DNT1 was predominantly secreted with a truncated prodomain (pro-DNT1), whereas DNT2 was secreted mature. In vivo, both pro- and mature DNTs were produced from neurons. Interestingly, DNT1 also has an isoform lacking the CK domain, and Spz has multiple isoforms with truncated prodomains. Thus, in vivo, whether DNT1 and 2 are secreted full length or cleaved and whether Spz is activated will depend on the proteases that each cell type may express. Pro-DNT1 activates apoptotic JNK signaling, whereas mature DNT1 and 2 activate the prosurvival NF-κB (Dorsal and Dif) and ERK signaling pathways. Mature Spz does not activate ERK. This first tier is evolutionarily conserved, as mammalian pro-NTs can promote cell death, whereas furin-cleaved mature NTs promote cell survival. NF-κB, JNK, and ERK are downstream targets shared with the mammalian NTs, downstream of p75NTR (NF-κB and JNK) and Trks (ERK), to regulate neuronal survival and death. Thus, whether a cell lives or dies will depend on the available proteases, the ligand type, and the ligand cleavage product it receives (Foldi, 2017).

    In a second regulatory tier, this study showed that the specific Toll family receptor activated by a DNT matters. Toll-6 and -7 could maintain neuronal survival, whereas Toll-1 had a predominant proapoptotic effect. Because there are nine Tolls in Drosophila, some Tolls could have prosurvival functions, whereas others could have proapoptotic functions. Different Tolls also lead to different cellular outcomes in immunity and development. Thus, the life or death of a neuron will depend on the Toll or combination of Tolls it expresses. Binding of Spz to Toll-1 is most likely unique, but DNT1 and 2 bind Toll-6 and -7 promiscuously, and, additionally, DNT1 and 2 with Toll-6 and -7 activate NF-κB and ERK, whereas pro-DNT1 activates JNK. This suggests that ligand prodomains might alter the affinity for Toll receptors and/or facilitate the formation of heterodimers between different Tolls and/or with other coreceptors to induce cell death. A 'DNT-Toll code' may regulate neuronal numbers (Foldi, 2017).

    In a third tier, available downstream adaptors determine the outcome between cell survival and death. Toll-6 and -7 activate cell survival by binding MyD88 and activating NF-κB and ERK (whether ERK activation depends on MyD88 is not known), and Toll-6 can activate cell death via Wek, dSarm, and JNK signaling. Toll-6 was shown to bind MyD88 and Wek, which binds dSarm, and dSarm binds MyD88 and promotes apoptosis by inhibiting MyD88 and activating JNK. Wek also binds MyD88 and Toll-1. So, evidence suggests that Wek recruits MyD88 and dSarm downstream of Tolls. Because Toll-6 binds both MyD88 and Wek and Wek binds both MyD88 and dSarm, Wek functions like a hinge downstream of Toll-6 to facilitate signaling via MyD88 or dSarm, resulting in alternative outcomes. Remarkably, adaptor expression profiles change over time, switching the response to Toll-6 from cell survival to cell death. In the embryo, when both MyD88 and dSarm are abundant, there is virtually no Wek, and Toll-6 can only bind MyD88 to promote cell survival. As Wek levels rise, Toll-6 signaling can also induce cell death. If the Wek-Sarm-JNK route prevails, Toll-6 induces apoptosis; if the Wek-MyD88-NF-κB route prevails, Toll-6 signaling induces cell survival (Foldi, 2017).

    Thus, the cellular outcome downstream of DNTs and Tolls is context and time dependent. Whether a cell survives or dies downstream of DNTs and Tolls will depend on which proteases are expressed nearby, which ligand it receives and in which form, which Toll or combination of Tolls it expresses, and which adaptors are available for signaling (Foldi, 2017).

    How adaptor profiles come about or change is not understood. A neuronal type may be born with a specific adaptor gene expression profile, or Toll receptor activation may influence their expression. In fact, MyD88 reinforces its own signaling pathway, as Toll-6 and -7 up-regulate Dorsal, Dif, and Cactus protein levels and TLR activation increases Sarm levels. This study showed that apoptosis caused by MyD88 excess depends on JNK signaling. Because JNK functions downstream of Wek and dSarm, this suggests that MyD88, presumably via NF-κB, can activate the expression of JNK, wek, or dsarm. By positively regulating wek expression, MyD88 and dSarm could establish positive feedback loops reinforcing their alternative pathways. Because dSarm inhibits MyD88, mutual regulation between them could drive negative feedback. Positive and negative feedback loops underlie pattern formation and structural homeostasis and could regulate neuronal number in the CNS as well. Whether cell-autonomous or -nonautonomous mechanisms result in the diversification of adaptor profiles, either in time or cell type, remains to be investigated (Foldi, 2017).

    Either way, over time the Toll adaptors segregate to distinct neural circuits, where they exert further functions in the CNS. Toll-1, -6, and -8 regulate synaptogenesis and structural synaptic plasticity. Sarm regulates neurite degeneration, and in the worm, it functions at the synapse to determine neuronal identity. The reporters used in this study revealed a potential segregation of MyD88 to the motor circuit and dSarm to the sensory circuit, but this is unlikely to reflect the endogenous complexity of Toll-signaling circuitry, as dsarmMIMIC- has a GFP insertion into one of eight potential isoforms, and dsarm also functions in the motor system. Importantly, cell death in the normal CNS occurs mostly in late embryogenesis and in pupae, coinciding with neural circuit formation and remodeling, when neuronal number is actively regulated. Thus, the link by DNTs and Tolls from cell number to circuitry offers a complex matrix of possible ways to regulate structural plasticity in the CNS (Foldi, 2017).

    This study has uncovered remarkable similarities between Drosophila Toll-6 and mammalian TLR signaling involving MyD88 and Sarm. All TLRs except TLR3 signal via MyD88 and activate NF-κB . Neuronal apoptosis downstream of TLRs is independent of NF-κB and instead depends on TRIF and Sarm1. Sarm1 is a negative regulator of TLR signaling, an inhibitor of MyD88 and TRIF. sarm1 is expressed in neurons, where it activates JNK and promotes apoptosis. However, the endogenous ligands for TLRs in the normal undamaged brains are not known. Preliminary analysis has revealed the intriguing possibility that NTs either can bind TLRs or induce interactions between Trks, p75NTR, and TLRs. It is compelling to find out whether TLRs regulate structural plasticity in the mammalian brain in concert with NTs (Foldi, 2017).

    To conclude, DNTs with Tolls constitute a novel molecular system for structural plasticity in the Drosophila CNS. This could be a general mechanism to be found also in the mammalian brain and in other contexts as well, such as epithelial cell competition and regeneration, and altered in cancer and neurodegeneration (Foldi, 2017).

    Nutraceutical Strategy to Counteract Eye Neurodegeneration and Oxidative Stress in Drosophila melanogaster Fed with High-Sugar Diet

    Aberrant production of reactive oxygen species (ROS) is a common feature of damaged retinal neurons in diabetic retinopathy, and antioxidants may exert both preventive and therapeutic action. To evaluate the beneficial and antioxidant properties of food supplementation with Lisosan G, a powder of bran and germ of grain (Triticum aestivum) obtained by fermentation with selected lactobacillus and natural yeast strains, an in vivo model was used of hyperglycemia-induced retinal damage, the fruit fly Drosophila melanogaster fed with high-sucrose diet. Lisosan G positively affected the visual system of hyperglycemic flies at structural/functional level, decreased apoptosis, and reactivated protective autophagy at the retina internal network. Also, in high sucrose-fed Drosophila, Lisosan G reduced the levels of brain ROS and retina peroxynitrite. The analysis of oxidative stress-related metabolites suggested key mediators of Lisosan G-induced inhibition of neuronal ROS, along with the upregulation of glutathione system. Of note, Lisosan G may impact oxidative stress and the ensuing retinal cell death, also independently from autophagy, although the autophagy-ROS cross-talk is critical. This study demonstrates that supplementation with Lisosan G exerts a antioxidant effect on retinal neurons, thus providing efficacious neuroprotection of hyperglycemic eye (Catalani, 2021).

    UQCRC1 engages cytochrome c for neuronal apoptotic cell death

    Human ubiquinol-cytochrome c reductase core protein 1 (UQCRC1) is an evolutionarily conserved core subunit of mitochondrial respiratory chain complex III. This study recently identified the disease-associated variants of UQCRC1 from patients with familial parkinsonism, but its function remains unclear. This study investigates the endogenous function of UQCRC1 in the human neuronal cell line and the Drosophila nervous system. Flies with neuronal knockdown of uqcrc1 exhibit age-dependent parkinsonism-resembling defects, including dopaminergic neuron reduction and locomotor decline, and are ameliorated by UQCRC1 expression. Lethality of uqcrc1-KO is also rescued by neuronally expressing UQCRC1, but not the disease-causing variant, providing a platform to discern the pathogenicity of this mutation. Furthermore, UQCRC1 associates with the apoptosis trigger cytochrome c (cyt-c), and uqcrc1 deficiency increases Cyt-c in the cytoplasmic fraction and activates the caspase cascade. Depleting cyt-c or expression of the anti-apoptotic p35 ameliorates uqcrc1-mediated neurodegeneration. The findings identified a role for UQCRC1 in regulating cyt-c-induced apoptosis (Hung, 2021).

    The appearance of cytoplasmic cytochrome C precedes apoptosis during Drosophila salivary gland degradation
    Apoptosis is an important process for organism development that functions to eliminate cell damage, maintain homeostasis, and remove obsolete tissues during morphogenesis. In mammals, apoptosis is accompanied by the release of cytochrome C (Cyt-c) from mitochondria to the cytoplasm. However, whether this process is conserved in the fruit fly, Drosophila melanogaster, remains controversial. This study discovered that during the degradation of Drosophila salivary gland, the transcription of mitochondria apoptosis factors (MAPFs), Cyt-c, and death-associated APAF1-related killer (Dark) encoding genes are all upregulated antecedent to initiator and effector caspases encoding genes. The proteins Cyt-c and the active caspase 3 appear gradually in the cytoplasm during salivary gland degradation. Meanwhile, the Cyt-c protein colocates with mito-GFP, the marker indicating cytoplasmic mitochondria, and the change in mitochondrial membrane potential coincides with the appearance of Cyt-c in the cytoplasm. Moreover, impeding or promoting 20E-induced transcription factor E93 suppresses or enhances the staining of Cyt-c and the active caspase 3 in the cytoplasm of salivary gland, and accordingly decreases or increases the mitochondrial membrane potential, respectively.This research provides evidence that cytoplasmic Cyt-c appears before apoptosis during Drosophila salivary gland degradation, shedding light on partial conserved mechanism in apoptosis between insects and mammals (Long, 2023).

    Acheron/Larp6 Is a Survival Protein That Protects Skeletal Muscle From Programmed Cell Death During Development

    programmed cell death (PCD) requires de novo gene expression. Using the ISMs from the tobacco hawkmoth Manduca sexta, this study has found that Acheron/LARP6 mRNA is induced ∼1,000-fold on the day the muscles become committed to die. Acheron functions as a survival protein that protects cells until cell death is initiated at eclosion (emergence), at which point it becomes phosphorylated and degraded in response to the peptide Eclosion Hormone (EH). Acheron binds to a novel BH3-only protein that was named BBH1 (BAD/BNIP3 homology 1/CG5059). BBH1 accumulates on the day the ISMs become committed to die and is presumably liberated when Acheron is degraded. This is correlated with the release and rapid degradation of cytochrome c and the subsequent demise of the cell. RNAi experiments in the fruit fly Drosophila confirmed that loss of Acheron results in precocious ecdysial muscle death while targeting BBH1 prevents death altogether. Acheron is highly expressed in neurons and muscles in humans and drives metastatic processes in some cancers, suggesting that it may represent a novel survival protein that protects terminally differentiated cells and some cancers from death (Sheel, 2020).

    Transiently "Undead" Enterocytes Mediate Homeostatic Tissue Turnover in the Adult Drosophila Midgut

    This study reveals surprising similarities between homeostatic cell turnover in adult Drosophila midguts and "undead" apoptosis-induced compensatory proliferation (AiP) in imaginal discs. During undead AiP, immortalized cells signal for AiP, allowing its analysis. Critical for undead AiP is the Myo1D-dependent localization of the initiator caspase Dronc to the plasma membrane. This study shows that Myo1D functions in mature enterocytes (ECs) to control mitotic activity of intestinal stem cells (ISCs). In Myo1D mutant midguts, many signaling events involved in AiP (ROS generation, hemocyte recruitment, and JNK signaling) are affected. Importantly, similar to AiP, Myo1D is required for membrane localization of Dronc in ECs. It is proposed that ECs destined to die transiently enter an undead-like state through Myo1D-dependent membrane localization of Dronc, which enables them to generate signals for ISC activity and their replacement. The concept of transiently "undead" cells may be relevant for other stem cell models in flies and mammals (Amcheslavsky, 2020).

    Rab21 in enterocytes participates in intestinal epithelium maintenance

    Membrane trafficking is defined as the vesicular transport of proteins into, out of, and throughout the cell. In intestinal enterocytes, defects in endocytic/recycling pathways result in impaired function and are linked to diseases. However, how these trafficking pathways regulate intestinal tissue homeostasis is poorly understood. Using the Drosophila intestine as an in vivo system, we investigated enterocyte-specific functions for the early endosomal machinery.This study focused on Rab21, which regulates specific steps in early endosomal trafficking. Depletion of Rab21 in enterocytes led to abnormalities in intestinal morphology, with deregulated cellular equilibrium associated with a gain in mitotic cells and increased cell death. Increases in apoptosis and Yorkie signaling were responsible for compensatory proliferation and tissue inflammation. Using an RNA interference screen, this study identified regulators of autophagy and membrane trafficking that phenocopied Rab21 knockdown. It was further shown that Rab21 knockdown-induced hyperplasia was rescued by inhibition of epidermal growth factor receptor signaling. Moreover, quantitative proteomics identified proteins affected by Rab21 depletion. Of these, changes were validated in apolipoprotein ApoLpp and the trehalose transporter Tret1-1, indicating roles for enterocyte Rab21 in lipid and carbohydrate homeostasis, respectively. These data shed light on an important role for early endosomal trafficking, and Rab21, in enterocyte-mediated intestinal epithelium maintenance (Nassari, 2022).

    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).

    Previous studies revealed that G9a is important for early embryogenesis and essential for viability in mice. G9a is also highly conserved among various metazoans including Drosophila, frogs (Xenopus tropicalis), fish (Danio rerio, Tetraodon nigroviridis, and Takifugu rubripes), and mammals. In Drosophila, although G9a is not essential for viability, the results of the present study suggest that G9a is conserved from the fly to mammals because of its importance in starvation stress tolerance, to which organisms are often exposed in the wild. This is also the first indication that epigenetic regulator-like G9a plays an essential role in the acquisition of starvation tolerance (An, 2017).

    In order to clarify the underlying mechanisms by which the dG9a null mutant is more susceptible to starvation stress, 'bottom up' approaches have been used. Non-targeted GC-MS-based and targeted LC-MS/MS-based metabolic profiling was performed to investigate changes in the metabolome due to the loss of dG9a. The results obtained from metabolic profiles showed that dG9a played important roles in maintaining energy homeostasis, the key factor for nutrient stress tolerance. dG9a modulated energy reservoirs including amino acid, trehalose, glycogen, and TAG levels during starvation via the autophagic process. One of the unique features of the adult dG9aRG5 mutant is its higher content of glycogen under non-starved normal conditions than that of the wild-type. A previous study reported that the deletion of G9a in mouse adipose tissues promotes adipogenesis and increases body weight (Wang, 2013). These findings and the present results suggest that dG9a is also responsible for the suppression of adipogenesis, similar to mammalian G9a. Further analyses are needed in order to clarify this point (An, 2017).

    The results of the present study also indicated that dG9a controlled starvation-induced autophagy by activating the expression of Atg8a; however, dG9a generally represses gene expression by dimethylating H3K9. Previous studies reported that histone and non-histone protein methylation by G9a either activated or inhibited gene expression. This study also found that the catalytic activity of dG9a was not required for the acquisition of starvation stress resistance by dG9a. This is consistent with the results of immunostaining showing that H3K9me2 levels in the nuclei of fat body cells under starvation were not significantly affected by the loss of dG9a. G9a has also been reported to activate gene expression as a molecular scaffold for the assembly of transcriptional co-activators, and the catalytic domain of G9a is not required for this function. Further studies are needed in order to clarify the mechanisms by which dG9a regulates the expression of Atg8a (An, 2017).

    Similar Atg8a mRNA levels were observed after 6h of fasting between wild-type and dG9aRG5 mutant flies; however, Atg8a immunostaining signals was weaker in the dG9aRG5 mutant than in the wild-type . Therefore, the loss of dG9a may repress the expression of genes that control Atg8a protein stability. Further studies are needed in order to elucidate the underlying mechanisms. During the development of Drosophila, metamorphosis is also a process that flies use to tolerate starvation stress. Even though this study demonstrated that dG9a is important for starvation stress tolerance, the viability of the dG9aRG5 mutant was not significantly less than that of the wild-type during the pupal stage. Together with the current results showing that the viability of the dG9aRG5 mutant at the larval stage was not affected by fasting conditions, the function of dG9a for starvation stress appears to be specific to the adult stage. Since programmed autophagy during the 3rd instar larval and pupal stages is well-known to be regulated by ecdysone through the PI3K pathway, starvation-induced autophagy by dG9a in the adult stage may be operated by other pathways (An, 2017).

    G9a is suggested to play a positive role in the promotion of tumorigenesis in various human cancer cells such as prostate, leukemia, lung, breast, and aggressive ovarian carcinoma. The inhibition of G9a activity in cancer cells significantly inhibited cell proliferation by triggering cell cycle arrest, inducing apoptosis, or activating autophagic cell death. The novel results obtained in this study on the role of dG9a to acquire starvation tolerance may also make it possible to explain the positive role of G9a in the promotion of tumorigenesis. Cells inside a tumor mass are exposed to starvation conditions because nutrients are not fully supplied to these cells. In order to overcome starvation stress, autophagy is induced in these cells. Therefore, G9a may play a role in the acquisition of starvation tolerance in cells in the tumor mass. The present study found that the loss of dG9a led to the inactivation of starvation-induced autophagy due to a decrease in Atg8a levels. In contrast, a previous study on cancer cells showed that the loss of G9a during starvation activated the transcription of LC3B (the Atg8a ortholog in mammals) and triggered autophagy (Martinez de Narvajas, 2013). Taken together, these results suggest that the epigenetic gene regulation of G9a depends on cell/tissue types (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).

    Misfolded protein aggregates in and outside of cells in the central nervous system are pathological hallmarks of many neurodegenerative disorders including Alzheimer (AD), Parkinson (PD), Huntington (HD) diseases and amyotrophic lateral sclerosis (ALS). Interestingly, many of the aggregated proteins (such as MAPT (TAU) and APP for Alzheimer disease, SNCA/α-synuclein for Parkinson disease, HTT (Huntingtin) for Huntington disease, FUS, SOD1 and TARDBP/TDP-43 for ALS) can serve as seeds for 'prion-like' spreading of the aggregation within and among cells. It is not entirely clear whether these aggregates are the causes or the results of progressive and cell-type-specific neurodegeneration. However, mounting evidence suggests that clearance and prevention of these toxic protein aggregates are beneficial for meliorating degeneration (Zhu, 2017).

    Two major pathways collaborate in regulating intracellular protein degradation: the ubiquitin-proteasome system (UPS) and the autophagy-lysosomal system. Under the normal conditions, UPS serves as the primary route for rapid protein turnover while autophagy mainly degrades long-lived proteins and large cellular organelles under basal conditions and can be robustly induced in face of stresses such as starvation, organelle damage or accumulation of misfolded proteins. However when it comes to degradation of damaged proteins in diseased states, autophagy has been shown to play at least an equally important role as UPS.5. Many of the neurodegenerative disease-related proteins are delivered to autophagic vacuoles and degraded by the autophagy pathway. Meanwhile, impairment of autophagy in the mouse brain causes neurodegeneration associated with ubiquitin-positive protein aggregation. These data suggest that UPS and autophagy are both indispensable in maintaining cellular protein homeostasis. Furthermore, recent studies indicate that UPS and autophagy pathways coordinate with each other to prevent accumulation of toxic protein aggregates, so that enhanced activity of one pathway can compensate if the other is compromised (Zhu, 2017).

    Both UPS and autophagy degradation systems are complex processes consisting of chains of sequential events orchestrated by a large group of proteins. To understand their coordinated action, it is necessary to identify novel players that are necessary and sufficient to mediate the compensatory function between the twi systems. This study shows Mask, a conserved protein with Ankyrin repeats and a KH domain, as a novel and critical player in such a context. Initially identified as a modulator of receptor tyrosine signaling during Drosophila development (Smith, 2002), Mask has recently been shown to function as a cofactor of the Hippo pathway effector Yorkie and together they regulate target gene transcription with another transcription cofactor (Scalloped) during cell proliferation (Sansores-Garcia, 2013; Sidor, 2013). The human ortholog of Mask, ANKHD1, is highly expressed in several cancer cell lines. Loss of mask function rescues the mitochondrial defects and muscle degeneration observed with pink1 and park mutants (Zhu, 2015). This study shows that in MAPT- and FUS-induced eye degeneration fly models, loss of Mask function enhances degeneration, while gain of Mask function suppresses degeneration. By enhancing V-type ATPase expression, Mask promotes lysosome acidification and autophagic flux; Mask is necessary and sufficient to mediate a compensatory effect for partial loss of UPS function, to increase clearance of ubiquitinated proteins, and to protect against degeneration induced by aggregation-prone mutations (Zhu, 2017).

    Autophagy, an evolutionarily conserved cellular mechanism that preserves metabolic homeostasis during nutrient unavailability, is traditionally regarded as a self-eating degradative process with limited selectivity. However, mounting evidence suggests that both micro- and macro-autophagy can play cytoprotective roles to specifically target damaged and toxic organelles and proteins for clearance under pathological conditions. The mechanism of selective autophagy is unclear. There is some evidence that autophagy receptors can recognize ubiquitin-dependent and ubiquitin-independent signals for selective degradation. Autophagy is a multistep process including nucleation, autophagosome formation and fusion with lysosomes and each step can be regulated to enhance degradation of damaged cellular components. Research has emerged showing TFEB is a potent regulator of the autophagy-lysosomal pathway whose activation can promote lysosomal function and mitigate disease in a range of neurodegenerative disorders. This study shows that Mask acts in a TFEB-independent manner to boost the expression of V-ATPase subunits. This study provides novel evidence that lysosome function is not only required for the normal clearance of ubiquitinated and misfolded proteins, but its activity can also be boosted potential through enhanced lysosomal acidification, to mitigate cellular degeneration caused by toxic protein aggregation (Zhu, 2017).

    Mask is well positioned to regulate lysosome-mediated clearance of ubiquitinated and misfolded proteins. As a positive regulator of several V-type ATPase V1 subunits expression, Mask function is necessary and sufficient to promote lysosomal acidification and autophagosome degradation in a cell-autonomous manner. When the UPS function is impaired, increased Mask expression is sufficient to increase autophagic flux, which in turn compensates the partial loss of the proteasome-mediated degradation. Interestingly, even when UPS function is intact, levels of Mask activity impact the abundance of UPS-dependent (K48) and -independent (such as K63) ubiquitin-conjugated proteins, suggesting that autophagy and lysosome-mediated degradation plays an important role for basal protein homeostasis. Under pathological conditions such as UPS inactivation or excessive accumulation of disease proteins, upregulation of Mask activity substantially suppressed the cellular degeneration phenotypes in both muscles and photoreceptors, potentially through Mask-mediated increase of autophagy and lysosome activities and subsequent degradation of harmful protein aggregates, as suggested by the current biochemical and genetic analyses. In support of this notion, upregulation of Mask promotes autophagic flux in larval muscles, adult eyes and adult brains (Zhu, 2017).

    This work in the Drosophila model organism yielded new insight into Mask-mediated cellular protective mechanisms that regulate lysosomal function in normal and stressed conditions caused by misfolding-prone disease proteins or impaired UPS. Such mechanisms may provide a therapeutic approach for the treatment of a group of neurodegenerative disorders caused by intracellular inclusions (Zhu, 2017).

    The FUS gene is dual-coding with both proteins contributing to FUS-mediated toxicity

    Novel functional coding sequences (altORFs) are camouflaged within annotated ones (CDS) in a different reading frame. This study shows that an altORF is nested in the FUS CDS, encoding a conserved 170 amino acid protein, altFUS. AltFUS is endogenously expressed in human tissues, notably in the motor cortex and motor neurons. Over-expression of wild-type FUS and/or amyotrophic lateral sclerosis-linked FUS mutants is known to trigger toxic mechanisms in different models. These include inhibition of autophagy, loss of mitochondrial potential and accumulation of cytoplasmic aggregates. altFUS, not FUS, is responsible for the inhibition of autophagy, and pivotal in mitochondrial potential loss and accumulation of cytoplasmic aggregates. Suppression of altFUS expression in a Drosophila model of FUS-related toxicity protects against neurodegeneration. Some mutations found in ALS patients are overlooked because of their synonymous effect on the FUS protein. Yet, this study shows they exert a deleterious effect causing missense mutations in the overlapping altFUS protein. These findings demonstrate that FUS is a bicistronic gene and suggests that both proteins, FUS and altFUS, cooperate in toxic mechanisms (Brunet, 2020).

    Multiple functions of the SNARE protein Snap29 in autophagy, endocytic, and exocytic trafficking during epithelial formation in Drosophila

    How autophagic degradation is linked to endosomal trafficking routes is little known. This study screened a collection of uncharacterized Drosophila mutants affecting membrane transport to identify new genes that also have a role in autophagy. A loss of function mutant was isolated in Snap29 (Synaptosomal-associated protein 29 kDa), the gene encoding the Drosophila homolog of the human protein SNAP29; and its function was characterized in vivo. Snap29 contains 2 soluble NSF attachment protein receptor (SNARE) domains and a asparagine-proline-phenylalanine (NPF motif) at its N terminus and rescue experiments indicate that both SNARE domains are required for function, whereas the NPF motif is in part dispensable. Snap29 was found to interact with SNARE proteins, localizes to multiple trafficking organelles, and is required for protein trafficking and for proper Golgi apparatus morphology. Developing tissue lacking Snap29 displays distinctive epithelial architecture defects and accumulates large amounts of autophagosomes, highlighting a major role of Snap29 in autophagy and secretion. Mutants for autophagy genes do not display epithelial architecture or secretion defects, suggesting that the these alterations of the Snap29 mutant are unlikely to be caused by the impairment of autophagy. In contrast, evidence was found of elevated levels of hop-Stat92E (hopscotch-signal transducer and activator of transcription protein at 92E) ligand, receptor, and associated signaling, which might underlie the epithelial defects. In summary, these findings support a role of Snap29 at key steps of membrane trafficking, and predict that signaling defects may contribute to the pathogenesis of cerebral dysgenesis, neuropathy, ichthyosis, and palmoplantar keratoderma (CEDNIK), a human congenital syndrome due to loss of Snap29 (Morelli, 2014).

    Organ development and homeostasis require concerted regulation of membrane trafficking routes, such as those governing protein secretion and endo-lysosomal degradation, and those controlling macroautophagy (autophagy hereafter), which regulates turnover of organelles and large cytoplasmic proteins. Studies in model organisms have clearly shown that the endo-lysosomal degradation pathway is required for correct organ development, due to its ability to promote degradation of signaling receptors controlling tissue growth and polarity. Such a major role of endocytosis on tissue architecture is underscored by the fact that Drosophila larval imaginal discs, a recognized model of epithelial organ development, when mutant for a number of the Endosomal Sorting Complexes Required for Transport (ESCRT) genes, display loss of polarity and overactivation of major signaling pathways, including N (Notch) and hop-Stat92E. In contrast, mutants in genes controlling autophagy often do not display loss of tissue architecture, or altered signaling phenotypes, indicating that impairment of endo-lysosomal or autophagic degradation have dramatically distinct consequences on tissue development. However, it is poorly understood which regulators of trafficking are required for formation and convergence of autophagosomes into the endosomal degradation route, and their relevance to organ development and homeostasis (Morelli, 2014).

    In autophagy, double-membrane organelles called autophagosomes are formed by a phagophore that sequesters portions of the cell cytoplasm. Autophagosomes then fuse with lysosomes, in which the autophagosome content is degraded. Studies have shown that 2 ubiquitin-like conjugation systems are required for autophagosome formation, and a number of organelles, such as the endoplasmic reticulum (ER), mitochondria, the Golgi apparatus, endosomes, and the plasma membrane have all been suggested to supply membranes and factors for autophagosome formation. Research in yeast indicates that, once formed, the autophagosome fuses with the vacuole, the yeast lysosome, in a manner dependent on the GTPase Ypt7/Rab7, on the homotypic fusion and protein sorting (HOPS) complex, and on SNARE-mediated membrane fusion. In metazoans, fusion events between autophagosomes and endosomal compartments are more complex, entailing the formation of amphisomes, which arise from fusion of autophagosomes with the multivesicular body (MVB), a late endosomal organelle. Consistent with this difference, in Drosophila and in mammalian cells ESCRT proteins, which regulate endosomal sorting and MVB formation, and the PtdIns3P 5-kinase fab1, which control endosome function, are required for amphisome and autolysosome formation. Also, differently from yeast, when formation of late endosomes is blocked in Drosophila and mammalian cells, autophagosomes accumulate in the cytoplasm, suggesting that amphisome formation helps clearance of autophagic cargoes (Morelli, 2014).

    The nature of SNARE-mediated fusion events occurring during formation and clearance of autophagosomes via the endo-lysosomal system is partly obscure. SNARE-mediated fusion involves a stereotypic set of SNARE proteins forming a 4-helix bundle composed by distinct SNARE domains named Qa-, Qb-, Qc- or R-SNARE. Usually, a Qa-SNARE-containing protein (a syntaxin, or t-SNARE) and a R-SNARE -containing protein (a VAMP protein, or v-SNARE) are carried by opposing membranes, and each provide a SNARE domain to the fusion complex. These proteins are glued together by Qb- and Qc- containing proteins, providing the remaining 2 SNARE domains. The Qb- and Qc-SNAREs involved in fusion events can be contributed by members of the SNAP protein family, with SNAP25 and SNAP23 being the most extensively studied. However, metazoan genomes also contain SNAP29, which, unlike other SNAP family members, contains a N-terminal NPF (asparagine-proline-phenylalanine) motif that binds endocytic adaptors, such as EDH1, and lacks palmitoylation sites for membrane anchoring. Consistent with this, SNAP29 resides in the cytoplasm and associates with membranes transiently. In contrast to its paralogs, SNAP29 has been much less studied and its function is unclear. In tissue culture and in in vitro studies, SNAP29 has been suggested to interact with multiple Qa-SNAREs such as syntaxins, and to associate with a number of intracellular organelles to promote-as well as inhibit-membrane fusion. Using depletion approaches, it has been shown that SNAP29 and its homolog in C. elegans and zebrafish regulates trafficking between several organelles, and that it is required for integrity of various intracellular compartments. Finally, in Drosophila and human cells, the SNAREs STX17/syntaxin 17 (Syx17) and vesicle-associated membrane protein 7 (VAMP7/Vamp7) have been very recently reported to act with SNAP29/Snap29 in fusion of autophagosomes to lysosomes (Takats, 2013; Itakura, 2012). Homozygous nonsense mutations leading to truncations of the human SNAP29 protein cause CEDNIK syndrome, a rare inherited congenital condition affecting skin and nervous system development and homeostasis, and resulting in short life span.32,33 Despite the evidence above, how SNAP29 functions and how its loss results in acquisition of CEDNIK traits is currently unclear (Morelli, 2014).

    This study used Drosophila imaginal discs to identify novel regulators of membrane trafficking that might have a role in autophagy, and to assess the importance of identified genes for epithelial organ development. With this strategy, the first Drosophila null mutant in Snap29 (also referred to as CG11173/usnp) was identified. Snap29 mutant imaginal discs present impairment of a late step of autophagy. In addition, it was found that Snap29 exerts an inhibitory role in membrane fusion at the apical membrane. In fact, Snap29 mutant tissue secretes autophagosomes in the apical lumen and presents excess of receptors on the plasma membrane. These defects correlate with disruption of the epithelial organization of imaginal discs and with a dramatic alteration in developmental signaling. Taken together, these data highlight a novel point of contact between trafficking and autophagy routes that is critical for organ development and might advance understanding of the CEDNIK pathogenesis (Morelli, 2014).

    The identity of SNARE proteins regulating the subsequent steps of fusion required for autophagosome formation and maturation into autolysosomes is a long-standing question, on which significant progress has been reported recently. The SNAREs STX12/STX13, Ykt6, Vamp7, and Sec22 have been recently proposed to be required for autophagosome formation in yeast, Drosophila and mammals. In yeast, the SNAREs Vam3, Vam7, and Vti1 have all been suggested to control fusion of autophagosomes with vacuoles. While Vam3 and Vam7 have no clear homologs in metazoan animals, the mammalian SNAREs VAMP7, VAMP8, and VTI1B are all suggested to be involved in autophagosomal fusion events. The Qa-SNARE protein STX17 is required for membrane fusion at 2 distinct steps of autophagy: Early autophagosome formation and fusion of autophagosomes with lysosomes to form autolysosomes. An association of Syx17 with Snap29 and the R-SNARE protein Vamp7 to form a fusion complex specific for late step of autophagy has been also very recently reported in the Drosophila fat tissue (Takats, 2013). Additionally, a certain degree of accumulation of autophagosomes has been observed in C. elegans depleted of Snap-29. Ultrastructural analysis showing clearly accumulation of almost exclusively fully formed autophagosomes with preserved luminal content strongly favors the model that Snap29 is required with Syx17 and Vamp7 for fusion of autophagosomes with lysosomes. Consistent with this evidence, accumulation of autophagosomes was found in Syx17 and Vamp7 mutant discs, and a genetic interaction was detected between Snap29 and Syx17, and Snap29 and Vamp7 (Morelli, 2014).

    An aspect that demands further investigation is whether Snap29 acts elsewhere in the endolysosomal system. Contrasting evidence was found for this. On one end, partial colocalization was found of Snap29 with the endosomal Qa-SNARE Syx7, and Syx7 was repeatedly found in immunoprecipitations. In addition, in uptake assays, in mutant cells the endocytic cargo N accumulates in an endosomal compartment. On the other end, such compartment is Syx7 negative. Since accumulation of N in a Syx7-positive endosomes has been reported to promote ectopic N activation, and this study has found reduced N signaling in Snap29 mutant discs, the point of N accumulation could be a postsorting compartment, such as the late endosome/MVB, or the lysosome. Despite this, no MVB accumulation was found in Snap29 mutant discs. These data are in sharp contrast with the accumulation of MVBs, but not of autophagosomes, that is observed in epithelial tissue mutant for vacuolar H+-ATPase (V-ATPase) subunit genes. Interestingly, in addition to enabling lysosomal functioning, V-ATPase have been proposed to play a role in membrane fusion and in autophagy. However, in addition to lack of accumulation of MVBs, very little sign is found of acid-induced degradation in the autophagosomes accumulated in Snap29 mutant cells. Thus, the comparison between the EM findings in Snap29 and V-ATPase mutants suggests that Snap29 functions upstream of V-ATPase in autophagy and argues against a role of V-ATPase in autophagosome formation or fusion to lysosomes (Morelli, 2014).

    Traits were observed in Snap29 mutant cells that could be the result of excess or inappropriate membrane fusion events, rather than of reduced fusion. These are: the large amount of membranes forming the accumulated autophagosomes; the presence in these of folded, multilamellar membranes; the secretion of autophagosomes extracellularly. It is unlikely that these events are an indirect result from the need of mutant cells to get rid of autophagic cargoes. In fact, no autophagosome secretion or excess membrane was found around autophagosomes in Syx17 and Vamp7 mutant discs. Alternatively, excess autophagosome membrane and secretion could both arise from failure to inhibit excess vesicle fusion. Inhibitory SNAREs have been postulated to occur naturally to control Golgi stack fusion patterns, while bacteria encode inhibitory SNAREs containing 2 SNARE domains, that can act with STX7 and VAMP8 (the homologs of Drosophila Syx7 and Vamp7) to inhibit secretion of lysosomes in mammalian cells. Interestingly, negative regulation of fusion by SNAP29 at the plasma membrane has been observed in rat neurons. A direct role of Snap29 in inhibition of membrane fusion at the plasma membrane during secretion could account also for the elevated N and dome levels on the surface of mutant cells. Consistent with this possibility, it was found that Snap29 interacts with Syx1A and Syx4, plasma membrane syntaxins and can localize to the plasma membrane upon overexpression. Of note, unconventional secretion routes involving autophagy regulators have been recently described, suggesting a scenario in which the autophagy and secretion functions of Snap29 could be connected to a putative negative role in fusion. The nature of Snap29 function in fusion events, and its involvement in unconventional secretion routes are currently under investigation (Morelli, 2014).

    Despite the large body of evidence on SNAP29, the pathogenesis of CEDNIK, a human congenital syndrome due to loss of Snap29, is obscure. Genetic analysis reveals that the Drosophila Snap29B6 mutant behaves as a strong loss of function and expresses a nonfunctional Snap29 protein, a similar situation to that reported for CEDNIK. Considering the absence of mouse mutants for Snap29, the findings in Drosophila could provide an initial framework to understand the pathogenesis of CEDNIK, which starts during fetal development and affects epithelial organs. In this regard, it was observed that the in vivo effect of lack of Snap29 during development in Drosophila is also epithelial tissue disorganization. This phenotype is unlikely to be due to impaired autophagy. In fact, genes specifically acting during autophagy, such as Atg13, Syx17, and Vamp7 were found to be dispensable for eye disc development. In addition, Atg7 appears dispensable for skin barrier formation in mice and flies. This evidence predicts that impairment of autophagy does not cause the developmental alterations associated to CEDNIK at least in the skin, which have been fairly well characterized. It is well possible that impaired autophagy plays a role in the unexplored neuronal traits of CEDNIK, considering that autophagy is a major process preventing neurodegeneration (Morelli, 2014)

    Which of the nonautophagy defects associated to lack of Snap29 could then be relevant to skin pathogenesis in CEDNIK? Could it be the defect highlighted by N accumulation in late endosomal and lysosomal compartments in an uptake experiment? This hypothesis is not favored. In fact, this study did not detect ectopic N activation, which is a feature of mutants of ESCRT genes controlling endosomal sorting. Such difference suggests that in Snap29 mutant cells, the pool of N accumulating intracellularly has been subjected to MVB sorting and resides in the late endosomal and lysosomal lumen. Considering also that loss of genes that control post MVB sorting events generally does not perturb disc epithelium development, the defect highlighted by intracellular N accumulation in Snap29 mutant cells is per se unlikely to contribute to the developmental phenotypes of Snap29 mutant organs (Morelli, 2014).

    Excluding routes that converge on the lysosomes, a further possibility is that the epithelial defects are due to alteration of secretory trafficking. Increased N presence at the plasma membrane, coupled with decreased N activation, could be relevant, since loss of N signaling is known to lead to epithelial alterations in skin. Alternatively, excess hop-Stat92E signaling could be important. In this case, excess signaling could directly originate from increased levels of active dome on the surface of Snap29 mutant cells. This scenario is consistent with the fact that Drosophila mutants preventing cargo internalization, such as those disrupting clathrin, display increased level of cargoes at the plasma membrane and possess elevated hop-Stat92E signaling and reduced N signaling. Underscoring a possible problem at the plasma membrane, expression of Socs36E, a negative regulator of hop-Stat92E signaling reported to act also by enhancing endosomal degradation of Dome, rescues part of the epithelial defects of Snap29 mutant discs. Alternatively, elevated Hop-Stat92E signaling could be a secondary effect of epithelial architecture or trafficking alterations. Detailed analysis of secretion and of signaling activity in CEDNIK samples will reveal whether alteration of these processes play a role in the pathogenesis of the syndrome (Morelli, 2014).

    In summary, this study clarifies the function of Snap29 in membrane trafficking and its consequences for epithelial tissue development, which might prove relevant for human health (Morelli, 2014).

    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).

    In a Drosophila model, neuronal necrosis was induced by the specific expression of a constitutively open glutamate receptor 1 channel (GluR1Lc) in neurons to overload calcium. By genetic screens using AG fly lethality, this study identified a novel suppressor of neuronal necrosis, LOF CG17259. CG17259 encodes a seryl-tRNA synthetase and functions in ligation of serine to its cognate tRNA. Therefore, LOF CG17259 may affect protein synthesis and induce cytoplasmic protein folding defects and/or ER stress. ER stress initiates through three distinct sensors in the ER membrane, including PERK, ATF6 and IRE1 (Deegan, 2013). Each signaling branch has both overlapping and distinct functions. For example, PERK phosphorylates eIF2α to reduce overall protein translation and promote cell survival. Whereas the IRE1 branch reduces protein synthesis by promoting the degradation of mRNA and activates JNK, which may, in turn, induce apoptosi. The current data demonstrated that the IRE1 branch was not activated in LOF CG17259, because transcription of Xbp1 sp and JNK pathway were not activated. In contrast, and the PERK/eIF2α branch was up-regulated in the LOF CG17259 flies. Consistent with these data, activation of the PERK/eIF2α signaling branch has been implicated in the treatment of various neurodegenerative diseases. For instance, treatment with salubrinal, an inhibitor of eIF2α dephosphorylation, can rescue neurodegeneration in α-synuclein transgenic mice or ischemic stroke in rats. Further, this study found that autophagy was activated in LOF CG17259. The coupling of the PERK/eIF2α signaling branch with autophagy has been well documented to protect neurons (Herz, 2014). The current research is consistent with these results from the literature. Additionally, this research provides an additional mechanism by which the eIF2α signaling pathway affects neuron survival (Lei, 2017).

    The results showed that the rescue effect of CG17259 −/+ was abolished by the mutants of Hsp26/Hsp27, and overexpression of Hsp26 or Hsp27 was sufficient to rescue AG flies, suggesting Hsp26/Hsp27 are down stream of LOF CG17259. The small chaperones of Drosophila Hsp26/Hsp27 are likely to have a similar function to that of mammalian Hsp27, which is known to protect neurons under various pathological conditions, including ischemic stroke. The protective mechanisms of Hsp27 may involve the suppression of the formation of actin aggregates, activation of the NF-κB pathway, or direct inhibition of components in the apoptotic machinery. The mammalian Hsp27 may share the combined function of Drosophila Hsp26/Hsp27, because it localizes in both cytosol and nucleus upon phosphorylation; while, it mainly localizes in the nucleus upon dephosphorylation. The current data showed that the Drosophila Hsp26 and Hsp27 distributed in cytosol or nucleus, respectively. For functional study, these data suggest that Hsp26/Hsp27 and p53 may function in the same pathway, because the rescue effect of p53 and CG17259 −/+ was not additive and Hsp26/Hsp27 protein could pull down p53. Although the co-IP data was obtained under the Hsp26/Hsp27 overexpression condition, the interaction between Hsp26/Hsp27 and p53 has been reported by other studies (Lei, 2017).

    The autophagy pathways can be further classified into autophagy (in this text macroautophagy refers to autophagy) and chaperone-mediated autophagy (CMA). Autophagy requires the formation of autophagosomes and the function of Atg genes. In contrast, the CMA pathway degrades proteins in lysosomes and does not require Atg genes (Todde, 2009). The current data suggested that autophagy was activated in the LOF CG17259 flies; up-regulation of autophagy rescued the AG lethality and down-regulation of autophagy had the opposite effect. Because LOF p53 rescued the enhancing death effect of LOF autophagy, it is possible that degradation of accumulated p53 was dependent on autophagy in the AG flies. Consistent with these data, the increase in the level of p53 protein has been observed in embryonic fibroblasts in Atg7 −/− or Atg5 −/− mice (Lei, 2017).

    Function of p53 in apoptosis has been well documented. Upregulation of p53 has been linked to neuronal cell death in numerous models of injuries and diseases, including excitotoxicity. The absence of p53 protects neurons from a wide variety of toxic insults, including focal ischemia, ionizing radiation and MPTP-induced neurotoxicity. In response to various types of stress, p53 promotes apoptosis through either transactivation of specific target genes or transcription-independent pathways. As a transcription factor, p53 upregulates proapoptotic genes, such as Bax, Noxa and PUMA. In addition, p53 can interact with Bcl2 family proteins, such as Bax and Bak, to induce permeabilization of the outer mitochondrial membrane. Whether p53 is involved in neuronal necrosis is unclear. In support of its involvement in necrosis, p53 may physically interact with cyclophilin D (CypD), a component of the mitochondrial permeability transition pores and trigger the opening of the pores and necrosis. In addition, the formation of the p53-CypD complex occurs during brain ischemia/reperfusion insult. This study provides the genetic and cell biology evidence indicating that p53 is involved in neuronal necrosis. In SH-SY5Y cells, it was shown that p53 was accumulated upon cells treated with glutamate; and this accumulation was prohibited by TM treatment, which enhanced Hsp27 transcription. Similarly, the increased level of p53 in MCAO rat brain was down-regulated by TM treatment. Together, these results indicate conserved function of p53 in neuronal necrosis. In fact, protective effect of TM against neurodegeneration has been widely reported. The difference is that the current study evaluated potential down-stream function of TM to degrade p53 in neuronal necrosis. How does p53 trigger both apoptosis and necrosis? It is proposed that mild p53 accumulation likely induces apoptosis, whereas the additional accumulation of p53 promotes necrosis. This hypothesis requires further investigation however (Lei, 2017).

    The inhibition of p53 transcriptional activity by pifithrin α or its mitochondrial targeting by pifithrin μ protects the brain in rodent models of stroke. However, p53 also benefits animal survival under hypoxic conditions. Thus, administration of pifithrins may interfere with the normal function of p53 and thereby produce side effects. An alternative way to target p53 may be to aim to reduce the accumulation of p53. This research suggests that the promotion of eIF2α signaling may activate endogenous mechanisms (activation of small chaperones and autophagy) to degrade p53 (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).

    Autophagy, one of the main degradative pathways of the cell, begins with the formation of a membranous cistern called phagophore or isolation membrane that buds from a cup-shaped structure associated with the endoplasmic reticulum (ER) called omegasome. Thereafter, the phagophore expands and finally seals, giving rise to a double membrane organelle named autophagosome where cytoplasmic components including protein aggregates, ribosomes, and mitochondria are sequestered. Soon afterward, autophagosomes acquire degradative enzymes by successive fusion with late endosomes and lysosomes, thereby becoming an autophagolysosome where the engulfed material is degraded (Melani, 2017).

    Autophagy, whose main stimulus is the stress generated by nutrient deprivation, is modulated by intracellular signaling pathways, mainly the target of rapamycin (TOR) and AMP-activated protein kinase (AMPK) cascades, as well as by extracellular factors including hormones. Activation of the ULK1 complex (Atg1 complex in yeast and Drosophila) has been described as the first event in the autophagy cascade. This complex, formed by ULK1/2, FIP200/Atg17, Atg13, and Atg101, is constitutively assembled, and its kinase activity is negatively regulated by TOR signaling, which in turn depends on amino acid availability and the energy status of the cell. ULK1/Atg1 regulates the recruitment and activation of a second complex: the Vps34 lipid kinase complex, also called the autophagy nucleation complex, which is composed of the class 3 phosphatidylinositol 3-kinase Vps34 and the proteins PI3KR4 (Vps15), Beclin1 (BECN1)/Atg6, and Atg14 (Melani, 2017).

    Vps34 mediates the synthesis of phosphatidylinositol 3-phosphate (PI3P). Local synthesis of this lipid defines the location of omegasome formation and, therefore the site of recruitment of several FYVE domain-containing proteins including DFCP1 and WIPI1, which in turn mediate phagophore elongation and autophagosome formation. Within the Vps34 complex, BECN1 is a direct target of ULK1/Atg1, and Vps34 kinase activity is believed to depend on the differential interaction of BECN1 with AMBRA1 or with the anti-apoptotic protein BCL-2. BCL-2 binding modulates the levels of BECN1 that become available to interact with Vps34 in the autophagy nucleation complex, thereby contributing to define if the cell will enter apoptosis or activate autophagy (Melani, 2017).

    FK506-binding proteins (FKBPs) play a role in immunoregulation and participate in critical cellular functions that include protein trafficking and folding. Members of this family display peptidyl prolyl cis/trans isomerase (PPIase) activity, participating in de novo protein folding through the interconversion of intermediate folding states into the final tridimensional structure. This study has investigated a novel Drosophila gene-which has been named Zonda (Zda)-that encodes an immunophilin of the FKBP family, presumably homologous to mammalian FKBP8/FKBP38 (Bhujabal, 2017; Melani, 2017 and references therein).

    By utilizing an in vivo approach, this study found that Zda is critically required for starvation-induced autophagy. Zda protein displays a cytoplasmic distribution in well-fed larvae and, shortly after the onset of starvation, nucleates in foci that colocalize with omegasome markers. Genetic manipulations revealed that components of the induction complex, Atg1, Atg13, and Atg17, but not components of the Vps34 complex, Vps34, Vps15, Atg6, or Atg14, are required for starvation-induced Zda nucleation. Moreover, Zda interacts physically with Atg1, Atg6, and Vps34 and is necessary for autophagic activation of Vps34 and omegasome formation, as revealed by DFCP1 foci formation following starvation. Zonda overexpression is sufficient to trigger a bona fide autophagic response, as evaluated by different autophagic markers. It is proposed that Zda is a novel component of the Drosophila autophagy machinery that forms part of the omegasome and is required for deposition of PI3P by the Vps34 complex and, hence, for the initiation of autophagosome biogenesis (Melani, 2017).

    Previously, other immunophilins have been proposed both as positive or negative regulators of autophagy. In Drosophila, FKBP39 was found to be a negative regulator of developmentally triggered autophagy, possibly through the regulation of the transcription factor Foxo. Mammalian FKBP51 was described as a scaffold protein that recruits PHLPP, Akt, and Beclin1, leading to activation of autophagy. FKBP38 has been reported as a mitophagy receptor that interacts with LC3. Coexpression of FKBP38 along with LC3 can trigger Parkin-independent mitophagy (Melani, 2017).

    Based on sequence homology, Zda is the likely orthologue of FKBP38. Not only do they share characteristics domains of FKBP proteins, but both proteins are the only members of their families to have a transmembrane domain on their C-terminal end. This study has shown that Zda is required for starvation-induced autophagy. Larval fat body cells in which Zda expression has been silenced fail to trigger autophagy, as assessed by several independent criteria: 1) inability of the cells to form autophagosomes and autolysosomes after starvation, as assessed by TEM and Atg8 nucleation; 2) their inability to increase the number and size of lysosomes, as evaluated by LysoTracker and GFP-Lamp markers; and 3) accumulation of Ref(2)P in these cells, which is indicative of impaired autophagic flux (Melani, 2017).

    This study has found that, after nutrient deprivation, Zda can be detected in omegasomes, colocalizing with PI3P and DFCP1, from which early autophagic structures labeled with GFP-Atg5 and GFP-Atg8 bud off. Consistent with the notion that Zda is an early component of the autophagy cascade, genetic analysis revealed that starvation-induced Zda nucleation depends fully on components of the Atg1 induction complex but not on components of the Vps34 nucleation complex. Vps34 autophagic activation following starvation is regulated by the nutritional status of the cell downstream of Atg1. This study found that Zda interacts physically with the Atg1 kinase domain, as well as with components of the nucleation complex, including Atg6 and Vps34, suggesting that it may contribute to the activation of the latter complex by Atg1. This notion is consistent with the results of genetic experiments utilizing early autophagy markers, as they suggest that autophagy-dependent Vps34 activation and omegasome formation are dependent on Zda, this dependence being comparable to that on Atg1. Unlike Atg6, which was shown to be also required for Vps34 basal activity, Zda is clearly not necessary for early endosome formation but only for autophagic activation of Vps34. Thus, given the requirement of Zda for Vps34 autophagy-specific activation, and based on its localization at the omegasome, it is proposed that Zda contributes to define the location on the ER at which Vps34-dependent PI3P deposition and omegasome formation take place (Melani, 2017).

    Induction of autophagy depends on the nutritional status of the cell and is subject to a contra-regulatory mechanism that occurs between mTOR and Atg1. Under nutrient-rich conditions, active mTOR phosphorylates and inactivates the Atg1 complex, and when nutrients are scarce, mTOR-dependent inactivation of Atg1 is released. Atg1 in turn reinforces down-regulation of mTOR through mechanisms that remain poorly defined. In line with this, Drosophila fat body cells that are mutant for atg1 grow bigger than control cells when subjected to prolonged nutrient deprivation, and conversely, Atg1 overexpression provokes cell size reduction and induces autophagosome formation. This study has shown that when overexpressed above certain levels, Zda can trigger a bona fide autophagic process, as assessed by several indicators, including TEM, Atg8 nucleation, and LysoTracker incorporation. This autophagic response fully depends on the activity of Vps34 and partially on Atg1. This suggests that Zonda operates upstream of Vps34 and in parallel to Atg1. Consistent with this, it was observed that under the same overexpression conditions, the TOR pathway is down-regulated and cell size is reduced similarly to what has been reported for Atg1. In line with these observations, adult flies that are homozygous for a Zda null mutation specifically in the head exhibit larger heads. Thus Zda mediates negative regulation of TOR, thereby exerting cell-­autonomous negative regulation of growth (Melani, 2017).

    Given that immunophilins are known to work as chaperons or scaffolds, it is proposed that Zda might provide a platform where Atg1 and the Vps34 complex interact. Further research is required to define the mechanism by which Zda cooperates with Atg1 on the activation of the Vps34 nucleation complex that culminates in localized PI3P deposition for omegasome formation (Melani, 2017).

    PtdIns4P exchange at endoplasmic reticulum-autolysosome contacts is essential for autophagy and neuronal homeostasis

    Inter-organelle contacts enable crosstalk among organelles, facilitating the exchange of materials and coordination of cellular events. This study demonstrated that, upon starvation, autolysosomes recruit Pi4KIIα (Phosphatidylinositol 4-kinase II α) to generate phosphatidylinositol-4-phosphate (PtdIns4P) on their surface and establish endoplasmic reticulum (ER)-autolysosome contacts through PtdIns4P binding proteins Osbp (Oxysterol binding protein) and cert (ceramide transfer protein). Sac1 (Sac1 phosphatase), Osbp, and cert proteins are required for the reduction of PtdIns4P on autolysosomes. Loss of any of these proteins leads to defective macroautophagy/autophagy and neurodegeneration. Osbp, cert, and Sac1 are required for ER-Golgi contacts in fed cells. These data establishes a new mode of organelle contact formation - the ER-Golgi contact machinery can be reused by ER-autolysosome contacts by re-locating PtdIns4P from the Golgi apparatus to autolysosomes when faced withstarvation (Liu, 2023).

    Characterization of the Autophagy related gene-8a (Atg8a) promoter in Drosophila melanogaster

    Autophagy is an evolutionarily conserved process which is upregulated under various stress conditions, including nutrient stress and oxidative stress. Amongst autophagy related genes (Atgs), Atg8a (LC3 in mammals) is induced several-fold during nutrient limitation in Drosophila. The minimal Atg8a cis-regulatory module (CRM) which mediates transcriptional upregulation under various stress conditions is not known. This study describes the generation and analyses of a series of Atg8a promoter deletions which drive the expression of an mCherry-Atg8a fusion cassette. Expression studies revealed that a 200 bp region of Atg8a is sufficient to drive expression of Atg8a in nutrient rich conditions in fat body and ovaries, as well as under nutrient deficient conditions in the fat body. Furthermore, this 200 bp region can mediate Atg8a upregulation during developmental histolysis of the larval fat body and under oxidative stress conditions induced by H2O2. Finally, the expression levels of Atg8a from this promoter are sufficient to rescue the lethality of the Atg8a mutant. The 200 bp promoter-fusion reporter provides a valuable tool which can be used in genetic screens to identify transcriptional and post-transcriptional regulators of Atg8a (Bali, 2017).

    A yeast two-hybrid screening identifies novel Atg8a interactors in Drosophila

    Macroautophagy/autophagy-related protein Atg8/LC3 is important for autophagosome biogenesis and required for selective degradation of various substrates. In a recent study, a yeast two-hybrid screening was performed to identify proteins that interact with Atg8a, the Drosophila homolog of Atg8/LC3. The screening identified several Atg8a-interacting proteins. These proteins include: i) proteins which have already been experimentally verified to bind Atg8a, such as Atg1, DOR, ref(2)P and key (Kenny); ii) proteins for which their mammalian homologs interact with Atg8-family members, like Ank2, Atg4, and Nedd4; and iii) several novel Atg8a-interacting proteins, such as trc/STK38 and Tak1. We showed that Tak1, as well as its co-activator, Tab2, both interact with Atg8a and are substrates for selective autophagic clearance. It was also determined that SH3PX1 interacts with Tab2 and is necessary for the effective regulation of the immune-deficiency (IMD) pathway. These findings suggest a mechanism for the regulatory interactions between Tak1-Tab2-SH3PX1 and Atg8a, which contribute to the fine-tuning of the IMD pathway (Tsapras, 2022b).

    Inflammation-induced, STING-dependent autophagy restricts Zika virus infection in the Drosophila brain

    The emerging arthropod-borne flavivirus Zika virus (ZIKV) is associated with neurological complications. Innate immunity is essential for the control of virus infection, but the innate immune mechanisms that impact viral infection of neurons remain poorly defined. Using the genetically tractable Drosophila system, this study shows that ZIKV infection of the adult fly brain leads to NF-kappaB-dependent inflammatory signaling, which serves to limit infection. ZIKV-dependent NF-kappaB activation induces the expression of Drosophila stimulator of interferon genes (dSTING) in the brain. dSTING protects against ZIKV by inducing autophagy in the brain. Loss of autophagy leads to increased ZIKV infection of the brain and death of the infected fly, while pharmacological activation of autophagy is protective. These data suggest an essential role for an inflammation-dependent STING pathway in the control of neuronal infection and a conserved role for STING in antimicrobial autophagy, which may represent an ancestral function for this essential innate immune sensor (Liu, 2018).

    The autophagy-related gene Atg101 in Drosophila regulates both neuron and midgut homeostasis

    Atg101 is an autophagy-related gene identified in worms, flies, mice, and mammals and encodes a protein that functions in autophagosome formation by associating with the ULK1-Atg13-Fip200 complex. Atg101's physiological role both during development and in adulthood remains poorly understood. This study describes the generation and characterization of an Atg101 loss-of-function mutant in Drosophila and reports on the roles of Atg101 in maintaining tissue homeostasis in both adult brains and midguts. Homozygous or hemizygous Atg101 mutants were semi-lethal, with only some of them surviving as adults. Both developmental and starvation-induced autophagy processes were defective in the Atg101 mutant animals, and Atg101 mutant adult flies had a significantly shorter lifespan and displayed a mobility defect. Moreover, accumulation of ubiquitin-positive aggregates were observed in Atg101 mutant brains, indicating a neuronal defect. Interestingly, Atg101 mutant adult midguts were shorter and thicker and exhibited abnormal morphology with enlarged enterocytes. Detailed analysis also revealed that the differentiation from intestinal stem cells to enterocytes was impaired in these midguts. Cell type-specific rescue experiments disclosed that Atg101 had a function in enterocytes and limited their growth. In summary, the results of this study indicate that Drosophila Atg101 is essential for tissue homeostasis in both adult brains and midguts. It is proposed that Atg101 may have a role in age-related processes (Guo, 2019).

    Crosstalk between Dpp and Tor signaling coordinates autophagy-dependent midgut degradation

    The majority of developmentally programmed cell death (PCD) is mediated by caspase-dependent apoptosis; however, additional modalities, including autophagy-dependent cell death, have important spatiotemporally restricted functions. Autophagy involves the engulfment of cytoplasmic components in a double membrane vesicle for delivery to the lysosome. An established model for autophagy-dependent PCD is Drosophila larval midgut removal during metamorphosis. Previous work demonstrated that growth arrest is required to initiate autophagy-dependent midgut degradation and Target of rapamycin (Tor) limits autophagy induction. This study uncovered a role for Decapentaplegic (Dpp) in coordinating midgut degradation. This study provides new data to show that Dpp interacts with Tor during midgut degradation. Inhibiting Tor rescued the block in midgut degradation due to Dpp signaling. It is proposed that Dpp is upstream of Tor and down-regulation promotes growth arrest and autophagy-dependent midgut degradation. These findings underscore a relationship between Dpp and Tor signaling in the regulation of cell growth and tissue removal (Denton, 2019).

    Control of basal autophagy rate by vacuolar peduncle

    Basal autophagy is as a compressive catabolic mechanism engaged in the breakdown of damaged macromolecules and organelles leading to the recycling of elementary nutrients. Thought essential to cellular refreshing, little is known about the origin of a constitutional rate of basal autophagy. This study found that loss of Drosophila vacuolar peduncle (vap), a presumed GAP enzyme, is associated with enhanced basal autophagy rate and physiological alterations resulting in a wasteful cell energy balance, a hallmark of overactive autophagy. By contrast, starvation-induced autophagy was disrupted in vap mutant conditions, leading to a block of maturation into autolysosomes. This phenotype stem for exacerbated biogenesis of PI(3)P-dependent endomembranes, including autophagosome membranes and ectopic fusions of vesicles. These findings shed new light on the neurodegenerative phenotype found associated to mutant vap adult brains in a former study. A partner of Vap, Sprint (Spri), acting as an endocytic GEF for Rab5, had the converse effect of leading to a reduction in PI(3)P-dependent endomembrane formation in mutants. Spri was conditional to normal basal autophagy and instrumental to the starvation-sensitivity phenotype specific of vap. Rab5 activity itself was essential for PI(3)P and for pre-autophagosome structures formation. It is proposed that Vap/Spri complexes promote a cell surface-derived flow of endocytic Rab5-containing vesicles, the traffic of which is crucial for the implementation of a basal autophagy rate (Bourouis, 2019).

    Autophagy within the mushroom body protects from synapse aging in a non-cell autonomous manner

    Macroautophagy is an evolutionarily conserved cellular maintenance program, meant to protect the brain from premature aging and neurodegeneration. How neuronal autophagy, usually loosing efficacy with age, intersects with neuronal processes mediating brain maintenance remains to be explored. This study shows that impairing autophagy in the Drosophila learning center (mushroom body, MB) but not in other brain regions triggered changes normally restricted to aged brains: impaired associative olfactory memory as well as a brain-wide ultrastructural increase of presynaptic active zones (metaplasticity), a state non-compatible with memory formation. Mechanistically, decreasing autophagy within the MBs reduced expression of an NPY-family neuropeptide, and interfering with autocrine NPY signaling of the MBs provoked similar brain-wide metaplastic changes. The results in an exemplary fashion show that autophagy-regulated signaling emanating from a higher brain integration center can execute high-level control over other brain regions to steer life-strategy decisions such as whether or not to form memories (Bhukel, 2019).

    The maintenance of neuronal homeostasis is severely threatened by aging. The strictly postnatal character of deficits observed after KD of core autophagy machinery triggered the hope that autophagy might have a specific relation to the aging process. The last few years have indeed seen an accumulation of evidences that the efficiency of autophagic clearance in neurons declines with age on organismal level. Hence, rejuvenating autophagy in aging neurons is considered a promising strategy to restore cognitive performance. Successfully exploring this direction will, however, depend on deepening insights at the intersection of autophagy, the relevant neuronal sub-cellular compartments, importantly synaptic specializations, and relevant neuron populations/brain regions (Bhukel, 2019).

    The endogenous polyamine spermidine has prominent cardio-protective and neuro-protective effects and recent work finds spermidine restoration to counteract otherwise deteriorating health in aging mice in an autophagy-dependent manner. In Drosophila, restoring spermidine specifically suppressed age-induced decay in their ability to form olfactory memories, again in an autophagy-dependent manner. Concomitantly, in the aged Drosophila brain, previous work found a brain-wide, age-induced upshift in the ultrastructural size (EM: larger T-bars; STED: increased diameter of BRP scaffold) of presynaptic AZs (metaplasticity). Two findings causally linked this upshift to decreased olfactory memory performance. First, when continuously fed with spermidine, flies of 30 days of age (normally suffering from a complete loss of age-sensitive component of memory) were largely protected from these changes. Secondly, genetically provoking this up-shift eliminated the normally age-sensitive memory component in young animals already. An upshift in the AZ size should increase synaptic strength, evident in increased SV release in response to natural odors observed in aged but not aged-spermidine-fed flies. Presynaptic plasticity is crucial for forming memory traces in Drosophila. Previous work thus suggests that this presynaptic metaplasticity shifts the operational range of synapses in a way that they become unable to execute the plastic changes faithfully in response to conditioning stimuli (Bhukel, 2019).

    This study further addressed the relation between defective autophagy, presynaptic ultrastructure and plasticity and olfactory memory formation. Autophagosome biogenesis is very dominant close to presynaptic specializations in distal axons in compartmentalized fashion and efficient macro-autophagy is essential for neuronal homeostasis and survival. Retrograde transport of autophagosomes might play a role in broader neuronal signaling processes, promoting neuronal complexity and preventing neurodegeneration. Surprisingly, however, the data do not favor a direct substrate relationship between AZ proteins and autophagy. Instead, evidence was found for a seemingly non-cell autonomous relation between brain-wide synapse organization and the autophagic status of the mere MB. After genetic impairment of autophagy (via atg5 or atg9 KD) using two different MB-specific Gal4-driver lines, the presynaptic metaplasticity was observed across the Drosophila olfactory system and beyond. While the autophagic arrest (p62 staining) was largely limited to the expression domain of these drivers, the synapses were pushed towards a state of metaplasticity. Since the ultrastructural size of AZs and the per AZ BRP levels increased equally in aged and MB-autophagy-challenged animals, it is concluded that the autophagic status of the MB neuron population executes a signaling process, which can control the per AZ amounts of BRP and other AZ proteins. Further studies are warranted to dissect the nature of these signaling processes (Bhukel, 2019).

    Notably, accumulating evidences support the important role of neuropeptide Y (NPY) in aging and lifespan determination. NPY levels decrease with age in mice and re-substituting NPY is able to counteract age-induced changes of the brain at several levels. A cross-talk between autophagy and NPY in regulating the feeding behavior has been demonstrated in mice (Bhukel, 2019).

    This study found that transcript expression level of an NPY family member (sNPF) are controlled by autophagy within the MBs. snpf hypomorph allele mimicking the MB reduction of sNPF of the MB-specific autophagy KD situations as well as the sNPF expression in aged animals. In this hypomorph allele a similar up regulation was observed in BRP Nc82 signal. KD of the snpfr using an MB-specific driver drove the brain-wide metaplastic change even stronger than the sNPF hypomorph (obviously only partially affecting the sNPF-specific signaling). This scenario in ultrastructural detail resembled both the age-induced and MB-specific autophagy-KD-induced metaplasticity phenotypes. These results, therefore, support the essential role of MB in integrating the metabolic state of Drosophila in an autocrine fashion to modulate the presynaptic release scaffold state throughout the fly brain. The mechanistic basis of this exciting regulation warrants further investigation. Interestingly, elevated cAMP signaling is generally driving plasticity in Drosophila neurons, while sNPF signaling is meant to reduce cAMP and thus potentially might be able to reset plastic changes such as increased BRP levels. In apparent contradiction to sNPF signaling directly widely controlling metaplasticity is the finding that MB-specific KD of the sNPFR sufficed to increase BRP levels. At this moment, it can only be speculated as to why KD of sNPF-receptor also results in extended metaplastic changes. Potentially, sNPF-receptor signaling within the MB might be important to control sNPF secretion in a physiological manner via a quasi-autocrine mechanism (Bhukel, 2019).

    Intriguingly, the metaplastic state characterized both aged and MB-specific autophagy KD animals, and in both cases provoked a specific loss of the ASM component of memory. Notably, olfactory MTM measured in this study, are considered to be the direct precursor of olfactory LTM, which in turn have been shown to be energetically costly. Notably, autophagy and NPY signaling are prime candidate mechanisms for the therapy of age-induced cognitive processes (Bhukel, 2019).

    Recent research has uncovered several examples connecting autophagy and hormonal-type regulations interacting between organ systems in non-cell autonomous regimes. For instance, Atg18 acts non-cell autonomously both in neurons and in intestines to firstly, maintain the wild-type lifespan of C.elegans and secondly, to respond to the dietary restriction and DAF-2 longevity signals. Atg18 in chemosensory neurons and intestines acts in parallel and converges on unidentified neurons that secrete neuropeptides to mediate the influence of Daf-2 on C.elegans lifespan through the transcription factor DAF-16/FOXO in response to reduced IGF signaling. In Drosophila, neuronal up-regulation of AMPK induces autophagy, via up-regulation of Atg1 non-cell autonomously in intestines and slows intestinal aging and vice versa. Moreover, up-regulation of Atg1 in neurons extends lifespan and maintains intestinal homeostasis during aging and these inter-tissue effects of AMPK/Atg1 were linked to altered insulin-like signaling. On the contrary, this study found the insulin producing cells (IPCs) themselves to not mediate the observed metaplastic state, as neither the KD of atg9 nor the KD of snpfr in Pars intercerebralis had any impact on the synaptic status of these flies (Bhukel, 2019).

    Autophagy regulation is tightly connected to cellular energetics, nutrient recycling, and the maintenance of cellular energy status. The fruit fly can evaluate its metabolic state by integrating hunger and satiety signals at the very KC-to-MBON synapses in MB under control of dopaminergic neurons to control hunger-driven food-seeking behavior. At the same time, long-term memory encoding necessitates an increase in MB energy flux with dopamine signaling mediating this energy switch in the MB. In line with these findings, this study now provides a modeling basis to study these delicate relations in an exemplary fashion. Taken together, these data suggest that MB integrates the metabolic state of the flies via cross talk between autophagy and sNPF signaling with the decision whether to form memories or not and a block in this cross talk with aging gives rise to synaptic metaplasticity which initiates the age-induced memory impairment in Drosophila. It is tempting to speculate that the MB executes hierarchically, a high-level control integrating the metabolic and caloric situation with a life-strategy decision of whether or not to form mid-term memories (Bhukel, 2019).

    Generation and characterization of germline-specific autophagy and mitochondrial reactive oxygen species reporters in Drosophila

    Oogenesis is a fundamental process that forms the egg and, is crucial for the transmission of genetic information to the next generation. Drosophila oogenesis has been used extensively as a genetically tractable model to study organogenesis, niche-germline stem cell communication, and more recently reproductive aging including germline stem cell (GSC) aging. Autophagy, a lysosome-mediated degradation process, is implicated in gametogenesis and aging. However, there is a lack of genetic tools to study autophagy in the context of gametogenesis and GSC aging. This study describes the generation of three transgenic lines mcherry-Atg8a, GFP-Ref(2)P and mito-roGFP2-Orp1 (an H2O2 sensor) that are specifically expressed in the germline compartment including GSCs during Drosophila oogenesis. These transgenes are expressed from the nanos promoter and present a better alternative to UASp mediated overexpression of transgenes. These fluorescent reporters can be used to monitor and quantify autophagy, and the production of reactive oxygen species during oogenesis. These reporters provide a valuable tool that can be utilized in designing genetic screens to identify novel regulators of autophagy and redox homeostasis during oogenesis (Nilangekar, 2019).

    Upregulation of the autophagy adaptor p62/SQSTM1 prolongs health and lifespan in middle-aged Drosophila

    Autophagy, a lysosomal degradation pathway, plays crucial roles in health and disease. p62/SQSTM1 (hereafter p62) is an autophagy adaptor protein that can shuttle ubiquitinated cargo for autophagic degradation. This study shows that upregulating the Drosophila p62 homolog ref(2)P/dp62, starting in midlife, delays the onset of pathology and prolongs healthy lifespan. Midlife induction of dp62 improves proteostasis, in aged flies, in an autophagy-dependent manner. Previous studies have reported that p62 plays a role in mediating the clearance of dysfunctional mitochondria via mitophagy. However, the causal relationships between p62 expression, mitochondrial homeostasis, and aging remain largely unexplored. This study shows that upregulating dp62, in midlife, promotes mitochondrial fission, facilitates mitophagy, and improves mitochondrial function in aged flies. Finally, this study shows that mitochondrial fission is required for the anti-aging effects of midlife dp62 induction. These findings indicate that p62 represents a potential therapeutic target to counteract aging and prolong health in aged mammals (Aparicio, 2019).

    Activation of Nrf2 in Astrocytes Suppressed PD-Like Phenotypes via Antioxidant and Autophagy Pathways in Rat and Drosophila Models

    The oxidative-stress-induced impairment of autophagy plays a critical role in the pathogenesis of Parkinson's disease (PD). This study investigated whether the alteration of Nrf2 in astrocytes protected against 6-OHDA (6-hydroxydopamine)- and rotenone-induced PD-like phenotypes, using 6-OHDA-induced rat PD and rotenone-induced Drosophila PD models. In the PD rat model, Nrf2 expression was significantly higher in astrocytes than in neurons. CDDO-Me (CDDO methyl ester, an Nrf2 inducer) administration attenuated PD-like neurodegeneration mainly through Nrf2 activation in astrocytes by activating the antioxidant signaling pathway and enhancing autophagy in the substantia nigra and striatum. In the PD Drosophila model, the overexpression of Nrf2 in glial cells displayed more protective effects than such overexpression in neurons. Increased Nrf2 expression in glial cells significantly reduced oxidative stress and enhanced autophagy in the brain tissue. The administration of the Nrf2 inhibitor ML385 reduced the neuroprotective effect of Nrf2 through the inhibition of the antioxidant signaling pathway and autophagy pathway. The autophagy inhibitor 3-MA partially reduced the neuroprotective effect of Nrf2 through the inhibition of the autophagy pathway, but not the antioxidant signaling pathway. Moreover, Nrf2 knockdown caused neurodegeneration in flies. Treatment with CDDO-Me attenuated the Nrf2-knockdown-induced degeneration in the flies through the activation of the antioxidant signaling pathway and increased autophagy. An autophagy inducer, rapamycin, partially rescued the neurodegeneration in Nrf2-knockdown Drosophila by enhancing autophagy. These results indicate that the activation of the Nrf2-linked signaling pathways in glial cells plays an important neuroprotective role in PD models (Guo, 2021).

    Macros to Quantify Exosome Release and Autophagy at the Neuromuscular Junction of Drosophila Melanogaster

    Automatic quantification of image parameters is a powerful and necessary tool to explore and analyze crucial cell biological processes. This article describes two ImageJ/Fiji automated macros to approach the analysis of synaptic autophagy and exosome release from 2D confocal images. Emerging studies point out that exosome biogenesis and autophagy share molecular and organelle components. Indeed, the crosstalk between these two processes may be relevant for brain physiology, neuronal development, and the onset/progression of neurodegenerative disorders. In this context, this study studied the macros "Autophagoquant" and "Exoquant" to assess the quantification of autophagosomes and exosomes at the neuronal presynapse of the Neuromuscular Junction (NMJ) in Drosophila melanogaster using confocal microscopy images. The Drosophila NMJ is a valuable model for the study of synapse biology, autophagy, and exosome release. By use of Autophagoquant and Exoquant, researchers can have an unbiased, standardized, and rapid tool to analyze autophagy and exosomal release in Drosophila NMJ (Sanchez-Mirasierra, 2021).

    Progressive transcriptional changes in metabolic genes and altered fatbody homeostasis in Drosophila model of Huntington's disease

    Huntington's disease (HD) is an autosomal-dominant neurodegenerative disorder marked by progressive neuronal atrophy, particularly in striatum and cerebral cortex. Although predominant manifestations of the disease include loss in the triad of motor, cognitive and behavioral capabilities, metabolic dysfunction in patients and HD models are being increasingly recognized. Patients display progressive body weight loss, which aggravates the disease and leads to cachexia in the terminal stages. Using the Drosophila model of HD, it was earlier reported that diseased flies exhibit an atypical pattern of lipid gain and loss with progression along with exhibiting extensive mitochondrial dysfunction, impaired calcium homeostasis and heightened apoptosis in the fatbody. This study first monitored the structural changes that abdominal fatbody undergoes with disease progression. Further, the transcriptional changes of key metabolic genes in whole fly were checked as well as genes regulating mitochondrial function, apoptosis, autophagy and calcium homeostasis in the abdominal fatbody. Extensive alterations were found in whole-body and fatbody-specific transcriptional profile of the diseased flies, which was in consort with their stage-specific physiological state. Additionally, lysosome-mediated autophagy was assessed in the fatbody of diseased flies in order to ascertain the mechanisms contributing to fatbody atrophy at the terminal stage. Interestingly, elevated autophagy was found in fatbody of flies throughout disease progression. This study provides new insights into the effect on peripheral metabolism due to degeneration of neurons in the neurodegenerative disease, thereby discerns novel mechanisms leading to cachexia in diseased flies and advocates for the need of managing metabolic dysfunctions in HD (Singh, 2022).

    Autophagy is required for spermatogonial differentiation in the Drosophila testis

    Autophagy is a conserved, lysosome-dependent catabolic process of eukaryotic cells which is involved in cellular differentiation. Its specific role in the differentiation of spermatogonial cells in the Drosophila testis was studied. In the apical part of the Drosophila testis, there is a niche of germline stem cells (GSCs), which are connected to hub cells. Hub cells emit a ligand for bone morhphogenetic protein (BMP)-mediated signalling that represses Bam (bag of marbles) expression in GSCs to maintain them in an undifferentiated state. GSCs divide asymmetrically, and one of the daughter cells differentiates into a gonialblast, which eventually generates a cluster of spermatogonia (SG) by mitoses. Bam is active in SG, and defects in Bam function arrest these cells at mitosis. This study shows that BMP signalling represses autophagy in GSCs, but upregulates the process in SG. Inhibiting autophagy in SG results in an overproliferating phenotype similar to that caused by bam mutations. Furthermore, Bam deficiency leads to a failure in downstream mechanisms of the autophagic breakdown. These results suggest that the BMP-Bam signalling axis regulates developmental autophagy in the Drosophila testis, and that acidic breakdown of cellular materials is required for spermatogonial differentiation (Varga, 2022).

    Inhibition of autophagy rescues muscle atrophy in a LGMDD2 Drosophila model

    Limb-girdle muscular dystrophy D2 (LGMDD2) is an ultrarare autosomal dominant myopathy caused by mutation of the normal stop codon of the TNPO3 nuclear importin. The mutant protein carries a 15 amino acid C-terminal extension associated with pathogenicity. This study reports the first animal model of the disease by expressing the human mutant TNPO3 gene in Drosophila musculature or motor neurons and concomitantly silencing the endogenous expression of the fly protein ortholog, Tnpo-SR. A similar genotype expressing wildtype TNPO3 served as a control. Phenotypes characterization revealed that mutant TNPO3 expression targeted at muscles or motor neurons caused LGMDD2-like phenotypes such as muscle degeneration and atrophy, and reduced locomotor ability. Notably, LGMDD2 mutation increase TNPO3 at the transcript and protein level in the Drosophila model. Upregulated muscle autophagy observed in LGMDD2 patients was also confirmed in the fly model, in which the anti-autophagic drug chloroquine was able to rescue histologic and functional phenotypes. Overall, this study provides a proof of concept of autophagy as a target to treat disease phenotypes, and a neurogenic component is proposed to explain mutant TNPO3 pathogenicity in diseased muscles (Blazquez-Bernal, 2021).

    HEXA-018, a Novel Inducer of Autophagy, Rescues TDP-43 Toxicity in Neuronal Cells

    The autophagy-lysosomal pathway is an essential cellular mechanism that degrades aggregated proteins and damaged cellular components to maintain cellular homeostasis. This study identified HEXA-018, a novel compound containing a catechol derivative structure, as a novel inducer of autophagy. HEXA-018 increased the LC3-I/II ratio, which indicates activation of autophagy. Consistent with this result, HEXA-018 effectively increased the numbers of autophagosomes and autolysosomes in neuronal cells. This study also found that the activation of autophagy by HEXA-018 is mediated by the AMPK-ULK1 pathway in an mTOR-independent manner. It was further shown that ubiquitin proteasome system impairment- or oxidative stress-induced neurotoxicity was significantly reduced by HEXA-018 treatment. Moreover, oxidative stress-induced mitochondrial dysfunction was strongly ameliorated by HEXA-018 treatment. In addition, the efficacy of HEXA-018 in models of TDP-43 proteinopathy was investigated. HEXA-018 treatment mitigated TDP-43 toxicity in cultured neuronal cell lines and Drosophila. These data indicate that HEXA-018 could be a new drug candidate for TDP-43-associated neurodegenerative diseases (Lee, 2021).

    Endophilin-B regulates autophagy during synapse development and neurodegeneration

    Synapses are critical for neuronal communication and brain function. To maintain neuronal homeostasis, synapses rely on autophagy. Autophagic alterations cause neurodegeneration and synaptic dysfunction is a feature in neurodegenerative diseases. In Parkinson's disease (PD), where the loss of synapses precedes dopaminergic neuron loss, various PD-causative proteins are involved in the regulation of autophagy. So far only a few factors regulating autophagy at the synapse have been identified and the molecular mechanisms underlying autophagy at the synapse is only partially understood. this study describes Endophilin-B (EndoB) as a novel player in the regulation of synaptic autophagy in health and disease. EndoB is required for autophagosome biogenesis at the synapse, whereas the loss of EndoB blocks the autophagy induction promoted by the PD mutation LRRK2(G2019S). EndoB is required to prevent neuronal loss. Moreover, loss of EndoB in the Drosophila visual system leads to an increase in synaptic contacts between photoreceptor terminals and their post-synaptic synapses. These data confirm the role of autophagy in synaptic contact formation and neuronal survival (Hernandez-Diaz, 2022).

    A new model for fatty acid hydroxylase-associated neurodegeneration reveals mitochondrial and autophagy abnormalities

    Fatty acid hydroxylase-associated neurodegeneration (FAHN) is a rare disease that exhibits brain modifications and motor dysfunctions in early childhood. The condition is caused by a homozygous or compound heterozygous mutation in fatty acid 2 hydroxylase (FA2H), whose encoded protein synthesizes 2-hydroxysphingolipids and 2-hydroxyglycosphingolipids and is therefore involved in sphingolipid metabolism. Drosophila is an excellent model for many neurodegenerative disorders; hence, this study has characterized and validated the first FAHN Drosophila model. The investigation of loss of dfa2h lines revealed behavioral abnormalities, including motor impairment and flying disability, in addition to a shortened lifespan. Furthermore, alterations in mitochondrial dynamics, and autophagy were identified. Analyses of patient-derived fibroblasts, and rescue experiments with human FA2H, indicated that these defects are evolutionarily conserved. This study thus presents a FAHN Drosophila model organism that provides new insights into the cellular mechanism of FAHN (Mandik, 2022).

    Autophagy controls Wolbachia infection upon bacterial damage and in aging Drosophila

    Autophagy is a conserved catabolic process in eukaryotic cells that degrades intracellular components in lysosomes, often in an organelle-specific selective manner (mitophagy, ERphagy, etc). Cells also use autophagy as a defense mechanism, eliminating intracellular pathogens via selective degradation known as xenophagy. Wolbachia pipientis is a Gram-negative intracellular bacterium, which is one of the most common parasites on Earth affecting approximately half of terrestrial arthropods. Interestingly, infection grants the host resistance against other pathogens and modulates lifespan, so this bacterium resembles an endosymbiont. This study demonstrates that Drosophila somatic cells normally degrade a subset of these bacterial cells, and autophagy is required for selective elimination of Wolbachia upon antibiotic damage. In line with these, Wolbachia overpopulates in autophagy-compromised animals during aging while its presence fails to affect host lifespan unlike in case of control flies. The autophagic degradation of Wolbachia thus represents a novel antibacterial mechanism that controls the propagation of this unique bacterium, behaving both as parasite and endosymbiont at the same time (Hargitai, 2023).

    Aging aggravates acetaminophen-induced acute liver injury and inflammation through inordinate C/EBPalpha-BMP9 crosstalk

    Previous studies have shown that bone morphogenetic protein 9 (BMP9) is almost exclusively produced in the liver and reaches tissues throughout the body as a secreted protein. However, the mechanism of BMP9 action and its role in aging-associated liver injury and inflammation are still unclear. Aging significantly aggravates acetaminophen (APAP)-induced acute liver injury (ALI). Increased expression of CCAAT/enhancer binding protein α (C/EBPα) and BMP9 was identified in aged livers and in hepatocytes and macrophages (MΦs) isolated from aged mice. Further analysis revealed that excess BMP9 was directly related to APAP-induced hepatocyte injury and death, as evidenced by activated Drosophila Mothers against decapentaplegic protein 1/5/9 (SMAD1/5/9) signaling, an increased dead cell/total cell ratio, decreased levels of ATG3 and ATG7, blocked autophagy, increased senescence-associated beta-galactosidase (SA-β-Gal) activity, and a higher rate of senescence-associated secretory phenotype (SASP) acquisition. In contrast, Bmp9 knockout (Bmp9(-/-)) partially alleviated the aforementioned manifestations of BMP9 overexpression. Moreover, BMP9 expression was found to be regulated by C/EBPα in vitro and in vivo. Notably, BMP9 also downregulated autophagy through its effect on autophagy-related genes (ATG3 and ATG7) in MΦs, which was associated with aggravated liver injury and SASP acquisition. In summary, the present study highlights the crucial roles played by C/EBPα-BMP9 crosstalk and provides insights into the interrelationship between hepatocytes and MΦs during acute liver injury (Liu, 2023).

    Codon-optimized TDP-43 mediates neurodegeneration in a Drosophila model of ALS/FTLD

    Transactive response DNA binding protein-43 (TDP-43) is known to mediate neurodegeneration associated with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). The exact mechanism by which TDP-43 exerts toxicity in the brains, spinal cord, and lower motor neurons of affected patients remains unclear. In a novel Drosophila melanogaster model, this study reports gain-of-function phenotypes due to misexpression of insect codon-optimized version of human wild-type TDP-43 (CO-TDP-43) using both the binary GAL4/UAS system and direct promoter fusion constructs. The CO-TDP-43 model showed robust tissue specific phenotypes in the adult eye, wing, and bristles in the notum. Compared to non-codon optimized transgenic flies, the CO-TDP-43 flies produced increased amount of high molecular weight protein, exhibited pathogenic phenotypes, and showed cytoplasmic aggregation with both nuclear and cytoplasmic expression of TDP-43. Further characterization of the adult retina showed a disruption in the morphology and function of the photoreceptor neurons with the presence of acidic vacuoles that are characteristic of autophagy. Based on these observations, it is proposed that TDP-43 has the propensity to form toxic protein aggregates via a gain-of-function mechanism, and such toxic overload leads to activation of protein degradation pathways such as autophagy. The novel codon optimized TDP-43 model is an excellent resource that could be used in genetic screens to identify and better understand the exact disease mechanism of TDP-43 proteinopathies and find potential therapeutic targets (Yusuff, 2023).

    PI3P-dependent regulation of cell size and autophagy by phosphatidylinositol 5-phosphate 4-kinase

    Phosphatidylinositol 3-phosphate (PI3P) and phosphatidylinositol 5-phosphate (PI5P) are low-abundance phosphoinositides crucial for key cellular events such as endosomal trafficking and autophagy. Phosphatidylinositol 5-phosphate 4-kinase (PIP4K) is an enzyme that regulates PI5P in vivo but can act on both PI5P and PI3P in vitro. This study repors a role for PIP4K in regulating PI3P levels in Drosophila Loss-of-function mutants of the only Drosophila PIP4K gene show reduced cell size in salivary glands. PI3P levels are elevated in dPIP4K 29 and reverting PI3P levels back towards WT, without changes in PI5P levels, can rescue the reduced cell size. dPIP4K 29 mutants also show up-regulation in autophagy and the reduced cell size can be reverted by depleting Atg8a that is required for autophagy. Lastly, increasing PI3P levels in WT can phenocopy the reduction in cell size and associated autophagy up-regulation seen in dPIP4K 29 Thus, this study reports a role for a PIP4K-regulated PI3P pool in the control of autophagy and cell size (Ghosh, 2023).

    Atg2 Regulates Cellular and Humoral Immunity in Drosophila

    Autophagy is a process that promotes the lysosomal degradation of cytoplasmic proteins and is highly conserved in eukaryotic organisms. Autophagy maintains homeostasis in organisms and regulates multiple developmental processes, and autophagy disruption is related to human diseases. However, the functional roles of autophagy in mediating innate immune responses are largely unknown. This study sought to understand how Atg2, an autophagy-related gene, functions in the innate immunity of Drosophila melanogaster. The results showed that a large number of melanotic nodules were produced upon inhibition of Atg2. In addition, inhibiting Atg2 suppressed the phagocytosis of latex beads, Staphylococcus aureus and Escherichia coli; the proportion of Nimrod C1 (one of the phagocytosis receptors)-positive hemocytes also decreased. Moreover, inhibiting Atg2 altered actin cytoskeleton patterns, showing longer filopodia but with decreased numbers of filopodia. The expression of AMP-encoding genes was altered by inhibiting Atg2. Drosomycin was upregulated, and the transcript levels of Attacin-A, Diptericin and Metchnikowin were decreased. Finally, the above alterations caused by the inhibition of Atg2 prevented flies from resisting invading pathogens, showing that flies with low expression of Atg2 were highly susceptible to Staphylococcus aureus and Erwinia carotovora carotovora 15 infections. In conclusion, Atg2 regulated both cellular and humoral innate immunity in Drosophila. This study has identified Atg2 as a crucial regulator in mediating the homeostasis of immunity, which further established the interactions between autophagy and innate immunity (Qin, 2023).

    Restoration of Sleep and Circadian Behavior by Autophagy Modulation in Huntington's Disease
    Circadian and sleep defects are well documented in Huntington's disease (HD). Modulation of the autophagy pathway has been shown to mitigate toxic effects of mutant Huntingtin (HTT) protein. However, it is not clear whether autophagy induction can also rescue circadian and sleep defects. Using a genetic approach, this study expressed human mutant HTT protein in a subset of Drosophila circadian neurons and sleep center neurons. In this context, the contribution was examined of autophagy in mitigating toxicity caused by mutant HTT protein. Targeted overexpression of an autophagy gene, Atg8a inmale flies, induces autophagy pathway and partially rescues several HTT-induced behavioral defects, including sleep fragmentation, a key hallmark of many neurodegenerative disorders. Using cellular markers and genetic approaches, it was demonstrated that indeed the autophagy pathway is involved in behavioral rescue. Surprisingly, despite behavioral rescue and evidence for the involvement of the autophagy pathway, the large visible aggregates of mutant HTT protein were not eliminated. The rescue in behavior is associated with increased mutant protein aggregation and possibly enhanced output from the targeted neurons, resulting in the strengthening of downstream circuits. Overall, this study suggests that, in the presence of mutant HTT protein, Atg8a induces autophagy and improves the functioning of circadian and sleep circuits (Sharma, 2023).

    Disrupted endoplasmic reticulum-mediated autophagosomal biogenesis in a Drosophila model of C9-ALS-FTD
    Macroautophagy/autophagy is a major pathway for the clearance of protein aggregates and damaged organelles, and multiple intracellular organelles participate in the process of autophagy, from autophagosome formation to maturation and degradation. Dysregulation of the autophagy pathway has been implicated in the pathogenesis of neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), however the mechanisms underlying autophagy impairment in these diseases are incompletely understood. Since the expansion of GGGGCC (G(4)C(2)) repeats in the first intron of the C9orf72 gene is the most common inherited cause of both ALS and FTD (C9-ALS-FTD), this study investigated autophagosome dynamics in Drosophila motor neurons expressing 30 G(4)C(2) repeats (30 R). In vivo imaging demonstrates that expression of expanded G(4)C(2) repeats markedly impairs biogenesis of autophagosomes at synaptic termini, whereas trafficking and maturation of axonal autophagosomes are unaffected. Motor neurons expressing 30 R display marked disruption in endoplasmic reticulum (ER) structure and dynamics in the soma, axons, and synapses. Disruption of ER morphology with mutations in Rtnl1 (Reticulon-like 1) or atl (atlastin) also impairs autophagosome formation in motor neurons, suggesting that ER integrity is critical for autophagosome formation. Furthermore, live imaging demonstrates that autophagosomes are generated from dynamic ER tubules at synaptic boutons, and this process fails to occur in a C9-ALS-FTD model. Together, these findings suggest that dynamic ER tubules are required for formation of autophagosomes at the neuromuscular junction, and that this process is disrupted by expanded G(4)C(2) repeats that cause ALS-FTD (Sung, 2023).

    A coherent FOXO3-SNAI2 feed-forward loop in autophagy

    Understanding autophagy regulation is instrumental in developing therapeutic interventions for autophagy-associated disease. This study identified SNAI2 as a regulator of autophagy from a genome-wide screen in HeLa cells. Upon energy stress, SNAI2 is transcriptionally activated by FOXO3 and interacts with FOXO3 to form a feed-forward regulatory loop to reinforce the expression of autophagy genes. Of note, SNAI2-increased FOXO3-DNA binding abrogates CRM1-dependent FOXO3 nuclear export, illuminating a pivotal role of DNA in the nuclear retention of nucleocytoplasmic shuttling proteins. Moreover, a dFoxO-Snail feed-forward loop regulates both autophagy and cell size in Drosophila, suggesting this evolutionarily conserved regulatory loop is engaged in more physiological activities (Guo, 2022).

    STING controls energy stress-induced autophagy and energy metabolism via STX17

    The stimulator of interferon genes (STING) plays a critical role in innate immunity. Emerging evidence suggests that STING is important for DNA or cGAMP-induced non-canonical autophagy, which is independent of a large part of canonical autophagy machineries. This study reports that, in the absence of STING, energy stress-induced autophagy is upregulated rather than downregulated. Depletion of STING in Drosophila fat cells enhances basal- and starvation-induced autophagic flux. During acute exercise, STING knockout mice show increased autophagy flux, exercise endurance, and altered glucose metabolism. Mechanistically, these observations could be explained by the STING-STX17 (Syntaxin 17) interaction. STING physically interacts with STX17, a SNARE that is essential for autophagosome biogenesis and autophagosome-lysosome fusion. Energy crisis and TBK1-mediated phosphorylation both disrupt the STING-STX17 interaction, allow different pools of STX17 to translocate to phagophores and mature autophagosomes, and promote autophagic flux. Taken together, this study demonstrates a heretofore unexpected function of STING in energy stress-induced autophagy through spatial regulation of autophagic SNARE STX17 (Rong, 2022).

    This study demonstrates the crucial role of STING in glucose metabolism through its negative regulation in energy stress-induced autophagy. The connection between autophagy and the cGAS-STING pathway has been intensively investigated with a focus on pathogens, DNA, or cyclic dinucleotides-induced autophagy through a non-canonical pathway. This study found that STING also plays a crucial role in energy stress-induced autophagy, especially in autophagosome-lysosome fusion through its interaction with the autophagic SNARE STX17. In the unstressed conditions, STING physically interacts with STX17 and sequesters it at ER. This interaction is disrupted by STING activation (DNA treatment etc) or autophagy stimuli (energy stress, etc.), which leads to STX17 translocation to autophagosomes, assembly of autophagic SNARE complex, and promotion of autophagosomal fusion with lysosomes. STING-regulated energy stress-induced autophagy has at least two effects, to facilitate elimination of DNA and microbes in immune cells and to boost energy metabolism in non-immune cells (Rong, 2022).

    STX17 is also important for autophagosome biogenesis. How the functions of STX17 in autophagy initiation and autophagosome-lysosome fusion is differentiated is an interesting question. It is proposed that STX17 phosphorylation by TBK1, as described by Kumar (2019), likely separates these two functions. TBK1-phosphorylated STX17 translocates from Golgi to mPAS, which is not controlled by STING, while the portion of STX17 that is not phosphorylated by TBK1 interacts with STING at ER/ERGIC (endoplasmic-reticulum-Golgi intermediate compartment), and this interaction is disrupted by autophagic stress likely through TBK1-independent regulatory events, which leads to translocation of this portion of STX17 from ER/ERGIC to complete autophagosomes. These observations nicely reconcile the different functions of STX17 in autophagy initiation and maturation (Rong, 2022).

    STING-regulated energy stress-induced autophagy is different from previously reported STING-mediated non-canonical autophagy in several aspects: (1) different membrane trafficking pathways are utilized. Triggered by PAMPs (pathogen-associated molecular patterns), STING translocates to single bilayer membrane vesicles positive for LC3, but these STING-LC3 positive vesicles are negative for STX17; (2) PAMPs-triggered STING-mediated autophagy is independent of BECN1, ULK1, and Atg9a. STX17 neither localizes to STING-positive vesicles nor is it required for STING trafficking and degradation; (3) PAMPs-induced non-canonical autophagy is compromised when STING is absent; while canonical autophagy is further activated in the absence of STING given that more STX17 is released from ER; (4) PAMPs-induced STING-dependent autophagy activation is limited to immune cells, but STING-regulated canonical autophagy functions broadly in both immune and non-immune cells (Rong, 2022).

    STING is a crucial regulator in the cancer-immunity cycle, and activation of STING represents a promising strategy for cancer therapy. This study suggests, in addition to immunity regulation, activation of STING also promotes energy stress-induced autophagy by releasing STX17 from ER. How autophagy activation contributes to STING mediates signaling remained to be investigated. At least, this study indicates that STING might play an unexpected broader role in energy metabolism due to its regulation of energy stress-induced autophagy. Autophagy has been implicated in a broad spectrum of human diseases, and STING also expresses and functions in non-immune tissues, suggesting that the regulatory effect of STING on autophagy might contribute to the pathogenesis of autophagy-related diseases and immune functions (Rong, 2022).

    Cyclin-G-associated kinase GAK/dAux regulates autophagy initiation via ULK1/Atg1 in glia

    Autophagy is a major means for the elimination of protein inclusions in neurons in neurodegenerative diseases such as Parkinson's disease (PD). Yet, the mechanism of autophagy in the other brain cell type, glia, is less well characterized and remains largely unknown. This study presents evidence that the PD risk factor, Cyclin-G-associated kinase (GAK)/Drosophila homolog Auxilin (dAux), is a component in glial autophagy. The lack of GAK/dAux increases the autophagosome number and size in adult fly glia and mouse microglia, and generally up-regulates levels of components in the initiation and PI3K class III complexes. GAK/dAux interacts with the master initiation regulator UNC-51-like autophagy activating kinase 1/Atg1 via its uncoating domain and regulates the trafficking of Atg1 and Atg9 to autophagosomes, hence controlling the onset of glial autophagy. On the other hand, lack of GAK/dAux impairs the autophagic flux and blocks substrate degradation, suggesting that GAK/dAux might play additional roles. Importantly, dAux contributes to PD-like symptoms including dopaminergic neurodegeneration and locomotor function in flies. These findings identify an autophagy factor in glia; considering the pivotal role of glia under pathological conditions, targeting glial autophagy is potentially a therapeutic strategy for PD (Zhang, 2023).

    Convergence of secretory, endosomal, and autophagic routes in trans-Golgi-associated lysosomes

    At the trans-Golgi, complex traffic connections exist to the endolysosomal system additional to the main Golgi-to-plasma membrane secretory route. This study investigated three hits in a Drosophila screen displaying secretory cargo accumulation in autophagic vesicles: ESCRT-III component Vps20, SNARE-binding Rop, and lysosomal pump subunit VhaPPA1-1. Vps20, Rop, and lysosomal markers were found to localize near the trans-Golgi. Furthermore, this study documented that the vicinity of the trans-Golgi is the main cellular location for lysosomes and that early, late, and recycling endosomes associate as well with a trans-Golgi-associated degradative compartment where basal microautophagy of secretory cargo and other materials occurs. Disruption of this compartment causes cargo accumulation in the hits, including Munc18 homolog Rop, required with Syx1 and Syx4 for Rab11-mediated endosomal recycling. Finally, besides basal microautophagy, it was shown that the trans-Golgi-associated degradative compartment contributes to the growth of autophagic vesicles in developmental and starvation-induced macroautophagy. These results argue that the fly trans-Golgi is the gravitational center of the whole endomembrane system (Zhao, 2023).

    A conserved interplay between FOXO and SNAI/snail in autophagy

    Dysfunction of macroautophagy/autophagy has been implicated in homeostasis maintenance and contributes to various diseases. Yet the mechanisms that regulate autophagy have not been fully understood. In a recent study, a coherent FOXO3-SNAI2 feed-forward regulatory loop in mammals was uncovered that reinforces autophagy gene induction upon energy stress. Strikingly, a foxo-sna (snail) feed-forward circuit also exists in Drosophila, suggesting this regulating loop is evolutionarily conserved. Moreover, these results highlight that binding of FOXO3 to the DNA appears to be both necessary and sufficient to antagonize CRM1-dependent nuclear export, illustrating a critical role of DNA in regulating protein nuclear localization (Guo, 2022).

    ER-mitochondrial contact protein Miga regulates autophagy through Atg14 and Uvrag

    Mitochondrial malfunction and autophagy defects are often concurrent phenomena associated with neurodegeneration. This study shows that Miga, a mitochondrial outer-membrane protein that regulates endoplasmic reticulum-mitochondrial contact sites (ERMCSs), is required for autophagy. Loss of Miga results in an accumulation of autophagy markers and substrates, whereas PI3P and Syx17 levels are reduced. Further experiments indicated that the fusion between autophagosomes and lysosomes is defective in Miga mutants. Miga binds to Atg14 and Uvrag; concordantly, Miga overexpression results in Atg14 and Uvrag recruitment to mitochondria. The heightened PI3K activity induced by Miga requires Uvrag, whereas Miga-mediated stabilization of Syx17 is dependent on Atg14. Miga-regulated ERMCSs are critical for PI3P formation but are not essential for the stabilization of Syx17. In summary, this study identified a mitochondrial protein that regulates autophagy by recruiting two alternative components of the PI3K complex present at the ERMCSs (Xu, 2022).

    Eukaryotic cells are compartmentalized into different organelles that execute distinct functions and communicate with each other through indirect signal transduction or direct organelle-organelle contacts. Mitochondria and the adjacent endoplasmic reticulum (ER) form contacts, which are characterized by a 10-30 nm distance between the two organelles. These contacts mediate lipid exchange and calcium flux between the ER and mitochondria. It has been reported that ER-mitochondrial contact sites (ERMCSs) are important platforms for regulating macroautophagy (hereafter referred to as autophagy) and mitophagy (Xu, 2022).

    Autophagosome formation at the ERMCSs in mammalian cells has been reported. Upon starvation, the ER-resident SNARE protein syntaxin 17 (STX17 in mammals; Syx17 in flies) recruits the PI3K complex subunit Atg14 to the ERMCSs and triggers autophagosome formation. However, Syx17 was not required for autophagosome formation in flies , and the major role of Syx17 in both mammals and flies is to mediate the fusion between autophagosome and lysosome. In addition, VAPB and PTPIP51, a pair of ERMCS tethers, also regulate autophagy. Increased ERMCS formation facilitated by VAPB or PTPIP51 overexpression inhibits autophagy; conversely, the weakening of contact by knockdown of these tethers stimulates autophagosome formation. Recent studies have shown that autophagy occurs at ERMCSs to supply free fatty acids for mitochondrial energy metabolism, while mitochondrial respiratory chain activity supports autophagy through the regulation of ERMCS formation. In addition to regulating autophagy at the initiation stage, in a previous study, it was determined that mitochondria play a crucial role in the late stage of autophagy. The loss of Tom40, a key subunit of the mitochondrial protein import channel, results in blockage of autophagosome and lysosome fusion. It was also found that defects in several general mitochondrial metabolic processes, such as ATP production, mitochondrial protein synthesis, or the citrate cycle, do not cause the autophagy defects observed in Tom40-depleted tissues. This implied that the autophagy defects caused by blocking mitochondrial protein import are rather specific. It is therefore hypothesized that certain mitochondrial proteins regulate autophagy directly (Xu, 2022).

    In the present study, it was demonstrated that Miga, a mitochondrial outer-membrane protein, is required for autophagy. Loss of Miga led to defects in autophagosome-lysosome fusion. Miga is an evolutionarily conserved protein, with orthologs from worms to humans. In a previous study, it was found to be localized on the mitochondrial outer membrane to regulate mitochondrial fusion by stabilizing MitoPLD. Miga interacts with the ER-localized VAP protein to establish ERMCSs. The interactions between Miga and VAP proteins are regulated by the phosphorylation of the FFAT motif in Miga. A recent study also reported that MIGA2 (the human ortholog of Miga) regulates ERMCSs and contacts between mitochondria and lipid droplets (LDs) . Loss of Miga led to the degeneration of photoreceptor cells in flies. Overexpression of Miga in fly eyes resulted in increased ERMCSs and severe eye degeneration. In mice, loss of MIGA2 led to anxiety-like behavior. This study found that Miga interacts with Atg14 and Uvrag to regulate PI3K activity and Syx17 stability, thereby modulating autophagy (Xu, 2022).

    Defects in both mitochondria and autophagy are hallmarks of several types of neurodegenerative diseases. This study found that Miga establishes a direct link between mitochondria and autophagy to maintain cellular homeostasis (Xu, 2022).

    It is striking that a mitochondrial protein directly regulates autophagy by interacting with the core components of the autophagy machinery. In the present study, it was found that the mitochondrial protein Miga forms complexes with Uvrag and Atg14 to regulate PI3P production and to stabilize Syx17 during autophagy (Xu, 2022).

    Miga interacts with Vap33 to mediate formation of ERMCSs. Overexpression of wild-type Miga, but not MigaFM, led to increased PI3P levels, implying that Miga-induced ERMCSs are required for regulating PI3P formation. However, the ERMCS tether function of Miga is neither required for recruiting Uvrag nor for binding to Atg14 and Syx17 stabilization. It has been shown previously that Atg14 and other components of the PI3K complex, such as Atg16 and Vps34, are enriched in ERMCSs upon starvation. The question remains as to why the PI3K complex needs to be present. Phosphatidylinositol (PI) is a substrate required for the PI3K complex to produce PI3P. PI is synthesized on the ER, and ERMCSs are the sites for the transfer of PI between the ER and mitochondria. During autophagy, the PI3K complex promotes PI3P formation to facilitate autophagic processes, and ERMCSs represent platforms to access PI. It is believed that the enrichment of the PI3K complex at ERMCSs is needed to assess the supply of PI. The present study found that MigaFM failed to promote PI3P formation, although it was still able to recruit key PI3K components, such as Uvrag or Atg14. This implied that PI3P formation during autophagy not only requires the activity of the PI3K complex but also PI supplied from ERMCSs (Xu, 2022).

    Previous studies reported that ERMCSs are required for the initiation of autophagy. In the current study, it was found that in Miga mutants, autophagic processes were blocked at the autophagosome-lysosome fusion stage, while autophagosome formation was largely unaffected. Lack of Miga led to a reduction in PI3P and Syx17 levels. Previous studies have demonstrated that PI3K is not only essential for autophagy initiation but is also recruited to the autophagosome together with the HOPS complex to facilitate autophagosome and lysosome fusion in mammalian cells. The remaining PI3P in Miga mutants is probably sufficient for autophagosome formation but not enough for the autophagosome-lysosome fusion process. This study found that the loss of Miga reduced co-localization of FYVE-GFP and Atg8a but not co-localization of FYVE-GFP and CathL. This suggests that the reduction of PI3P in autophagosomes, but not lysosomes, might contribute to fusion defects (Xu, 2022).

    The fusion defects observed in Miga mutants were not identical to those found in mutants without Syx17 or HOPS components. The puncta of autophagosome markers are larger in Miga mutants than those in mutants without Syx17 or HOPS components, possibly due to the combined effects of reduction of PI3P and Syx17. In worms and mammalian cells, the lack of EPG5 prevents autophagosome maturation and induces the ectopic fusion of autophagosomes with various endocytic vesicles. The enlarged Atg8a-positive structures in Miga mutants might also be a result of the ectopic fusion of autophagosomes with other vesicles (Xu, 2022).

    In mammals, both UVRAG and ATG14 are required for autophagy. In flies, Uvrag regulates PI3P formation under fed conditions, and Atg14 is required for PI3P-positive autophagosome formation. This study found that Miga overexpression induces PI3P formation; additionally, Uvrag, but not Atg14, is required during this process. It was also found that Miga overexpression leads to an upregulation of numerous autophagy markers, such as Atg9, Syx17, Atg18a, Rab7, and LAMP, among others. However, the expression levels or patterns of p62 and Atg8a did not change significantly upon Miga overexpression. This implied that Miga overexpression is not sufficient to fully activate autophagy (Xu, 2022).

    STX17, the mammalian ortholog of Syx17, is an autophagosome-localized Q-SNARE that mediates autophagosome and lysosome fusion through interactions with SNAP29 and VAMP8/Vamp7. STX17 contains two tandem transmembrane domains that have low hydrophobicity but are required for autophagosome localization. In fed mammalian cells, STX17 reportedly localizes to the ER, mitochondria, and cytosol. STX17 was enriched in ERMCSs upon autophagy stimulation and was present on completely closed autophagosomes. The detailed translocation mechanism remains unclear. In flies, Syx17 shows diffusely dispersed patterns, and there is no mitochondria-specific localization under normal fed conditions. Syx17 forms puncta and co-localizes with Atg8-positive autophagosomes upon starvation. It was found that Miga is required for the stabilization of Syx17. Miga does not bind to Syx17 but stabilizes it through Atg14. It is puzzling why a mitochondrial protein would be required for the stabilization of a protein that functions in autophagosome maturation. It has been reported that there are three-way contacts among the ER, mitochondria, and late endosomes. It is possible that Miga, Vap33, and Atg14 mediate the contact between the ER, mitochondria, and autophagosomes. Autophagosome-associated Atg14 further stabilizes Syx17 to mediate the fusion between autophagosomes and lysosomes. GFP-Atg14 formed large puncta instead of decreasing in the Miga mutant clones. One possible explanation for this is that the overexpression of GFP-Atg14 overrides the requirement of Miga to stabilize it, but the overexpression of GFP-Atg14 per se is not sufficient to fully rescue the autophagy defects in the Miga mutant. Therefore, similar to other autophagy markers, GFP-Atg14 puncta accumulated in the Miga mutant clones (Xu, 2022).

    In summary, this study identified a mitochondrial protein, Miga, that regulates autophagic processes by interacting with Atg14 and Uvrag. This delineates a link between mitochondria and macroautophagy. However, this study did not solve how Miga stabilizes Atg14 and Syx17. It is possible that Miga mediates the three-way contact between the ER, mitochondria, and autophagosomes. Miga interacts with Atg14 and stabilizes Atg14. Furthermore, Atg14 interacts with Syx17 to stabilize it. It is not clear how the relay is carried out during autophagy (Xu, 2022).

    Both Atg14 and Uvrag interact with MigaN (1-252 aa), but there is no evident competition between Atg14 and Uvrag. The exact regions of Miga that bind to each protein were not identified in this study (Xu, 2022).

    A conserved STRIPAK complex is required for autophagy in muscle tissue

    Autophagy is important for cellular homeostasis and to prevent the abnormal accumulation of proteins. While many proteins that comprise the canonical autophagy pathway have been characterized, the identification of new regulators may help understand tissue and/or stress-specific responses. Using an in-silico approach, Striatin interacting protein (Strip), MOB kinase activator 4, and fibroblast growth factor receptor 1 oncogene partner 2 were identified as conserved mediators of muscle tissue maintenance. Affinity purification-mass spectrometry (AP-MS) experiments with Drosophila melanogaster Strip was used as a bait protein and copurified additional Striatin-interacting phosphatase and kinase (STRIPAK) complex members from larval muscle tissue. NUAK family kinase 1 (NUAK) and Starvin (Stv) also emerged as Strip-binding proteins and these physical interactions were verified in vivo using proximity ligation assays. To understand the functional significance of the STRIPAK-NUAK-Stv complex, a sensitized genetic assay combined with RNA interference (RNAi) were used to demonstrate that both NUAK and stv function in the same biological process with genes that encode for STRIPAK complex proteins. RNAi-directed knockdown of Strip in muscle tissue led to the accumulation of ubiquitinated cargo, p62, and Autophagy-related 8a, consistent with a block in autophagy. Indeed, autophagic flux was decreased in Strip RNAi muscles, while lysosome biogenesis and activity were unaffected. These results support a model whereby the STRIPAK-NUAK-Stv complex coordinately regulates autophagy in muscle tissue (Guo, 2023).

    Upregulation of the autophagy adaptor p62/SQSTM1 prolongs health and lifespan in middle-aged Drosophila

    Autophagy, a lysosomal degradation pathway, plays crucial roles in health and disease. p62/SQSTM1 (hereafter p62) is an autophagy adaptor protein that can shuttle ubiquitinated cargo for autophagic degradation. This study shows that upregulating the Drosophila p62 homolog ref(2)P/dp62, starting in midlife, delays the onset of pathology and prolongs healthy lifespan. Midlife induction of dp62 improves proteostasis, in aged flies, in an autophagy-dependent manner. Previous studies have reported that p62 plays a role in mediating the clearance of dysfunctional mitochondria via mitophagy. However, the causal relationships between p62 expression, mitochondrial homeostasis, and aging remain largely unexplored. This study shows that upregulating dp62, in midlife, promotes mitochondrial fission, facilitates mitophagy, and improves mitochondrial function in aged flies. Finally, this study shows that mitochondrial fission is required for the anti-aging effects of midlife dp62 induction. These findings indicate that p62 represents a potential therapeutic target to counteract aging and prolong health in aged mammals (Aparicio, 2019).

    Loss of protein homeostasis (proteostasis) and mitochondrial dysfunction are two cellular hallmarks of aging, each of which has been proposed to contribute to age-related health decline. Therefore, identifying interventions that could improve proteostasis and/or mitochondrial function when targeted to aged animals could lead to treatments to forestall disease and promote healthy aging. Macroautophagy, hereafter autophagy, is a degradation pathway that plays key roles in development, tissue homeostasis, and disease pathogenesis. In this process, cellular materials (referred to as autophagic cargo) are sequestered by double-membrane vesicles known as autophagosomes (APs) and delivered to the lysosome for degradation. In recent years, autophagy, and more specifically a requirement for autophagy-related genes, has been implicated in genetic, dietary, and pharmacological interventions that extend lifespan in model organisms. These findings support the idea that autophagy induction plays a causal role in these prolongevity paradigms. In addition, constitutively increasing basal levels of autophagy, by directly manipulating autophagy-related genes, has been reported to promote longevity in diverse species including mice. Importantly, however, while autophagy induction is generally considered to be cytoprotective, it has also been linked to cell death and disease pathogenesis. Therefore, it is likely that in certain physiological contexts, autophagy can contribute to pathophysiology and, thereby, limit lifespan. Indeed, recent work has shown that inhibition of autophagy-related genes, in post-reproductive C. elegans, can prolong lifespan and health span, leading to the proposal that autophagy switches to a harmful role in aged animals. Hence, fundamental questions remain unanswered regarding the mechanism(s) by which age-related modulation of autophagy impacts organismal health and lifespan. Critically, there is a relative lack of understanding of how to modulate autophagy in aged animals to improve tissue homeostasis and prolong health (Aparicio, 2019).

    Autophagy receptors designate substrate specificity through the recognition of specific cargo, including protein aggregates (aggrephagy), mitochondria (mitophagy), and pathogens (xenophagy). Critically, however, an understanding of the role of autophagy cargo receptors in aging and lifespan determination remains elusive. p62 (also known as Sequestosome 1) is a prototypic autophagy adaptor that possesses a C-terminal ubiquitin-binding domain and a short LC3-interacting region responsible for LC3/ATG8 interaction, allowing recruitment of ubiquitinated cargo into nascent APs. Studies in Drosophila melanogaster and mice have shown that p62 is required for the aggregation of ubiquitinated proteins and their autophagic clearance. In addition, p62 has been shown to play a role in the PINK1/Parkin pathway of mitophagy. A key step in mitophagy involves the recruitment of Parkin, an E3 ubiquitin ligase, from the cytosol to a dysfunctional mitochondrion. Once there, Parkin ubiquitinates outer mitochondrial membrane proteins and induces mitophagy. p62 accumulates on damaged mitochondria and can recognize Parkin-mediated, poly-ubiquitinated chains (Geisler, 2010). The question of whether p62 is required for mitophagy remains controversial with some studies in mammalian cells reporting that p62 is required for Parkin-mediated mitophagy, but not others. Consistent with a key role for p62 in mitophagy, Refractory to Sigma P, ref(2)P, the single Drosophila ortholog of p62 has been shown to play an essential role in promoting mitophagy (de Castro, 2013). Studies in both flies and mice have shown that genetic inactivation of p62 leads to early-onset mitochondrial dysfunction, neurodegeneration, and reduced lifespan (de Castro, 2013; Kwon, 2012; Ramesh Babu, 2008). However, the consequences of increasing p62 expression, in aging animals, on mitochondrial homeostasis, proteotoxicity, and organismal health are not known (Aparicio, 2019).

    The proposed roles of p62 in the autophagic clearance of protein aggregates (aggrephagy) and dysfunctional mitochondria (mitophagy) led examination of whether p62 could modulate tissue and/or organismal aging. To do so, the impact of upregulation of the Drosophila p62 homolog ref(2)P/dp62 (de Castro, 2013) was examined at different stages of adulthood. Importantly, midlife induction of dp62 was shown to improves markers of health and prolongs lifespan. Short-term, midlife dp62 induction improves proteostasis in aged muscle. Critically, the ability of midlife dp62 induction to improve proteostasis and prolong lifespan is dependent upon autophagy-related genes. In addition, midlife dp62 induction promotes mitophagy and improves markers of mitochondrial function in aged flies. The process of mitophagy is intimately linked to mitochondrial fission/fusion processes. This study shows that inhibiting mitochondrial fission, via expression of a dominant-negative Dynamin-related protein 1 (Drp1) transgene, impairs dp62-mediated improvements in mitochondrial function and longevity. Furthermore, knockdown of parkin abrogates dp62-mediated longevity. Together, these findings indicate that activating dp62 expression, in midlife, is an effective approach to improve proteostasis and mitochondrial function and, thereby, prolong healthy lifespan (Aparicio, 2019).

    Numerous lines of evidence indicate that aging is linked to alterations in the activity of the autophagy pathway. However, the underlying mechanisms that lead to these changes and the causal relationships between altered autophagic activity and age-related health decline remain subject to speculation. Critically, the question of whether increasing the expression of autophagy-related genes in aged animals can slow tissue aging and/or promote longevity remains largely unexplored. This study used the fruit fly Drosophila as a model organism to address the question of whether p62, a prototypic autophagy cargo receptor, can modulate tissue and/or organismal aging. Using an inducible gene expression system, it was shown that upregulation of dp62 from midlife onward leads to a significant increase in fly lifespan. Furthermore, induction of dp62 in middle-aged animals improves several markers of organismal health and delays age-onset pathology. Induction of dp62 in young flies did not produce a prolongevity effect, indicating that dp62 expression levels are not limiting for health in early life. These findings reveal that p62 represents a therapeutic target to counteract aging and, thereby, prolong health span in aged animals. It is interesting to note that an 8-fold induction of dp62 mRNA levels was associated with lifespan extension (Aparicio, 2019).

    As P62 is a multifunctional protein that serves as a signaling hub for a myriad of cellular processes including amino acid sensing, immunity, and the oxidative response, there exist several potential candidate mechanisms that could underlie the beneficial effects of midlife dp62 induction. Critically, this study shows that the anti-aging effects of midlife dp62 induction, both at the tissue and organismal level, are dependent upon autophagy-related genes. More specifically, it was show that midlife dp62 induction reduces proteotoxicity in aged muscles and promotes longevity in an Atg1-dependent manner. Moreover, midlife dp62 induction leads to a shift toward mitochondrial fission and improves mitochondrial function in an Atg1-dependent fashion. To better understand the importance of mitochondrial fission in dp62-mediated longevity, this study set out to simultaneously inhibit mitochondrial fission and upregulate dp62 in midlife. Consistent with the idea that mitochondrial fission is important in facilitating mitophagy in aged animals, inhibiting mitochondrial fission abrogates the beneficial effects of midlife dp62 induction on mitochondrial function and longevity. Studies in yeast have shown that upon mitophagy induction, Dnm1 (yeast Drp1 ortholog) is recruited to the degrading mitochondria via the scaffold protein Atg11 to induce fission. Future work could focus on elucidating the molecular mechanisms by which increased dp62 expression promotes mitochondrial fission in aged animals. In addition, it was shown that dp62-mediated longevity requires parkin, a key component of the mitophagy pathway. These findings, therefore, indicate that the selective clearance of mitochondria, via mitophagy, is key to midlife dp62-mediated longevity (Aparicio, 2019).

    The autophagy pathway represents an attractive therapeutic target to promote healthy aging in humans. However, the question of when and how to manipulate autophagy in aging mammals, in order to prolong health, is not understood. A recent study reported that a gain-of-function mutation in a core autophagy gene, Becn1, can extend mammalian lifespan. However, it is not clear whether targeting approaches of this kind to aged mammals can promote longevity. Recent findings, in C. elegans, have shown that inhibiting genes involved in early stages of autophagy in aged animals can prolong lifespan. As a result, it has been proposed that dysfunctional autophagy in aged animals, linked to blockage of autophagy at a late stage, may contribute to age-onset health decline. Hence, it is possible that interventions that induce early stages of autophagy, including AP formation, may not promote health when targeted to aged animals. In contrast, the current findings suggest that midlife up-regulation of the autophagy adaptor protein, p62, can promote the autophagic clearance of protein aggregates and mitochondria in aged animals. Hence, increasing p62 expression by pharmacological means, in midlife, may be an effective approach to prolong health span in mammals (Aparicio, 2019).

    Tumor suppressive autophagy in intestinal stem cells controls gut homeostasis

    Re-routing intracellular vesicle traffic by suppressing macroautophagy/autophagy or endocytosis genes drastically deregulates Drosophila intestinal stem cell (ISC) proliferation, leading to massive gut hyperplasia that has a negative impact upon lifespan. Beginning with the poorly characterized Snx (sorting nexin) genes, this study surveyed a broad set of genes in the endocytosis-autophagy network and found that most of them have this effect. Deregulated Egfr-Ras85D/Ras1-mitogen-activated protein kinase signaling is the primary trigger for ISC proliferation upon disruption of this network; in the mutants, ligand-activated receptors were stabilized and recycled to the cell surface via Rab11-dependent endosomes, rather than being degraded via autophagosomes. This study profiled the mutational landscape for orthologous network genes in human cancers using The Cancer Genome Atlas (TCGA), and revealed strong, novel associations with distinct genomic and epigenomic subtypes of colorectal cancer (Zhang, 2019).

    Autophagy promotes tumor-like stem cell niche occupancy

    Adult stem cells usually reside in specialized niche microenvironments. Accumulating evidence indicates that competitive niche occupancy favors stem cells with oncogenic mutations, also known as tumor-like stem cells. However, the mechanisms that regulate tumor-like stem cell niche occupancy are largely unknown. This study used Drosophila ovarian germline stem cells as a model and use bam mutant cells as tumor-like stem cells. Interestingly, it was found that autophagy is low in wild-type stem cells but elevated in bam mutant stem cells. Significantly, autophagy is required for niche occupancy by bam mutant stem cells. Although loss of either atg6 or Fip200 alone in stem cells does not impact their competitiveness, loss of these conserved regulators of autophagy decreases bam mutant stem cell niche occupancy. In addition, starvation enhances the competition of bam mutant stem cells for niche occupancy in an autophagy-dependent manner. Of note, loss of autophagy slows the cell cycle of bam mutant stem cells and does not influence stem cell death. In contrast to canonical epithelial cell competition, loss of regulators of tissue growth, either the insulin receptor or cyclin-dependent kinase 2 function, influences the competition of bam mutant stem cells for niche occupancy. Additionally, autophagy promotes the tumor-like growth of bam mutant ovaries. Autophagy is known to be induced in a wide variety of tumors. Therefore, these results suggest that specifically targeting autophagy in tumor-like stem cells has potential as a therapeutic strategy (Zhao, 2018).

    This study used Drosophila ovarian germline stem cells to study stem cell competition for niche occupancy. Significantly, it was found that autophagy promotes niche occupancy by bam mutant stem cells. Autophagy is required for proper cell cycle of bam mutant cells, and regulators of growth influence bam mutant stem cell niche occupancy. Previous reports indicate that autophagy is required for stem cell maintenance, proper differentiation, and homeostasis in different stem cell systems. By contrast, the data indicate that loss of autophagy does not have a negative impact on Drosophila ovarian germline stem cells, consistent with data indicating that autophagy is low in normal wild-type stem cells. Drosophila ovarian germline stem cells are the largest cells in germaria, and they have a high metabolic rate that is associated with the activation of BMP signaling, the expression of Myc that is a key regulator of cell growth and ribosome biogenesis, and a high level of rRNA transcription. Therefore, the catabolic autophagy pathway may be dispensable in Drosophila ovarian germline stem cells under normal conditions (Zhao, 2018).

    Stem cell renewal is dependent on cell growth and division that is typically regulated by mTOR. In most cell contexts, autophagy is inhibited when mTOR-dependent cell growth is activated. Therefore, it is logical that autophagy levels are low in normal stem cells that are not stressed. By contrast, transformed cells, such as those with activated Ras, have been reported to possess elevated autophagy. Therefore, it seems reasonable that, like Ras transformed cells, bam mutant stem cells may depend on autophagy for cell division and ovarian growth. Interestingly, autophagy is required for the proliferation of fast-dividing germline progenitor cells in the C. elegans larval gonad. It is not clear why autophagy may be compatible with and required for tumor-like germline stem cell growth and division. One possibility is that autophagy is functioning to reduce cell stress associated with increased metabolic rate, protein, and organelle damage, but if this were the case, this would likely be reflected in increased cell death. Because non increase in cell death was observed, an alternative explanation is that autophagy is promoting bioenergetic homeostasis that is needed in tumor-like cells (Zhao, 2018).

    Unlike previous studies of epithelial cell competition, the data indicate that loss of regulators of tissue growth, either the insulin receptor or cyclin-dependent kinase 2 function, influence the competition of tumor-like stem cells for niche occupancy. Stem cells possess properties that distinguish them from epithelial cells in the context of cell competition. First, adult stem cells are usually quiescent, while epithelial cell competition often takes place in fast-growing tissues. Second, stem cells compete for niche occupancy and there are no known specialized niches in epithelial cell competition systems. Third, loser stem cells are displaced from the niche and do not die, while loser epithelial cells die, enabling winners to expand during epithelial cell competition. In addition, tumor-like stem cells appear to divide faster than normal adult stem cells. These differences probably make tumor-like stem cells more sensitive to regulators of growth than epithelial cells during competition (Zhao, 2018).

    Autophagy can either promote or suppress tumor growth, depending on cell and tissue context. Similar phenomena were also observed in different Drosophila tumor-like overgrowth models, where autophagy either enhanced or suppressed epithelial overgrowth phenotypes, depending on the oncogenic stimulus. The results indicate that autophagy promotes Drosophila ovarian germline tumor-like growth. Significantly, autophagy promotes niche occupancy by bam mutant tumor-like stem cells. Of note, the data indicate that the super-competition of bam mutant stem cells largely depends on their proliferative potential. In addition, the data contradict the current model that the niche stem cell adhesion factor E-cadherin plays a vital role, as no significant difference was observed in E-cadherin between wild-type and bam mutant stem cells. Furthermore, bam mutant stem cells that are out of the niche do not possess an E-cadherin connection with niche cap cells, but they are still more competitive than wild-type stem cells for niche occupancy, further challenging the current model that emphasizes a critical role of E-cadherin. In addition, although the super-competition of tumor-like stem cells for niche occupancy is proposed to be important for tumor initiation, it still cannot be excluded that autophagy also contributes to tumor progression in the tumor model system. Importantly, these studies indicate that specifically targeting autophagy in tumor-like stem cells could have potential for cancer therapy (Zhao, 2018).

    A tissue- and temporal-specific autophagic switch controls Drosophila pre-metamorphic nutritional checkpoints

    Properly timed production of steroid hormones by endocrine tissues regulates juvenile-to-adult transitions in both mammals (puberty) and holometabolous insects (metamorphosis). Nutritional conditions influence the temporal control of the transition, but the mechanisms responsible are ill defined. This study demonstrates that autophagy acts as an endocrine organ-specific, nutritionally regulated gating mechanism to help ensure productive metamorphosis in Drosophila. Autophagy in the endocrine organ is specifically stimulated by nutrient restriction at the early, but not the late, third-instar larva stage. The timing of autophagy induction correlates with the nutritional checkpoints, which inhibit precocious metamorphosis during nutrient restriction in undersized larvae. Suppression of autophagy causes dysregulated pupariation of starved larvae, which leads to pupal lethality, whereas forced autophagy induction results in developmental delay/arrest in well-fed animals. Induction of autophagy disrupts production of the steroid hormone ecdysone at the time of pupariation not by destruction of hormone biosynthetic capacity but rather by limiting the availability of the steroid hormone precursor cholesterol in the endocrine cells via a lipophagy mechanism. Interestingly, autophagy in the endocrine organ functions by interacting with the endolysosome system, yet shows multiple features not fully consistent with a canonical autophagy process. Taken together, these findings demonstrate an autophagy mechanism in endocrine cells that helps shape the nutritional checkpoints and guarantee a successful juvenile-to-adult transition in animals confronting nutritional stress (Pan, 2019).

    Autophagy-dependent filopodial kinetics restrict synaptic partner choice during Drosophila brain wiring

    Brain wiring is remarkably precise, yet most neurons readily form synapses with incorrect partners when given the opportunity. Dynamic axon-dendritic positioning can restrict synaptogenic encounters, but the spatiotemporal interaction kinetics and their regulation remain essentially unknown inside developing brains. This study shows that the kinetics of axonal filopodia restrict synapse formation and partner choice for neurons that are not otherwise prevented from making incorrect synapses. Using 4D imaging in developing Drosophila brains, this study shows that filopodial kinetics are regulated by autophagy, a prevalent degradation mechanism whose role in brain development remains poorly understood. With surprising specificity, autophagosomes form in synaptogenic filopodia, followed by filopodial collapse. Altered autophagic degradation of synaptic building material quantitatively regulates synapse formation as shown by computational modeling and genetic experiments. Increased filopodial stability enables incorrect synaptic partnerships. Hence, filopodial autophagy restricts inappropriate partner choice through a process of kinetic exclusion that critically contributes to wiring specificity (Kiral, 2020).

    Human ZKSCAN3 and Drosophila M1BP are functionally homologous transcription factors in autophagy regulation

    Autophagy is an essential cellular process that maintains homeostasis by recycling damaged organelles and nutrients during development and cellular stress. ZKSCAN3 is the sole identified master transcriptional repressor of autophagy in human cell lines. How ZKSCAN3 achieves autophagy repression at the mechanistic or organismal level however still remains to be elucidated. Furthermore, Zkscan3 knockout mice display no discernable autophagy-related phenotypes, suggesting that there may be substantial differences in the regulation of autophagy between normal tissues and tumor cell lines. This study demonstrates that vertebrate ZKSCAN3 and Drosophila M1BP are functionally homologous transcription factors in autophagy repression. Expression of ZKSCAN3 in Drosophila prevents premature autophagy onset due to loss of M1BP function and conversely, M1BP expression in human cells can prevent starvation-induced autophagy due to loss of nuclear ZKSCAN3 function. In Drosophila ZKSCAN3 binds genome-wide to sequences targeted by M1BP and transcriptionally regulates the majority of M1BP-controlled genes, demonstrating the evolutionary conservation of the transcriptional repression of autophagy. This study thus  allows the potential for transitioning the mechanisms, gene targets and plethora metabolic processes controlled by M1BP onto ZKSCAN3 and opens up Drosophila as a tool in studying the function of ZKSCAN3 in autophagy and tumourigenesis (Barthez, 2020).

    Defects of full-length dystrophin trigger retinal neuron damage and synapse alterations by disrupting functional autophagy

    Dystrophin (dys) mutations predispose Duchenne muscular disease (DMD) patients to brain and retinal complications. Although different dys variants, including long dys products, are expressed in the retina, their function is largely unknown. This study investigated the putative role of full-length dystrophin in the homeostasis of neuro-retina and its impact on synapsis stabilization and cell fate. Retinas of mdx mice, the most used DMD model which does not express the 427-KDa dys protein (Dp427), showed overlapped cell death and impaired autophagy. Apoptotic neurons in the outer plexiform/inner nuclear layer and the ganglion cell layer had an impaired autophagy with accumulated autophagosomes. The autophagy dysfunction localized at photoreceptor axonal terminals and bipolar, amacrine, and ganglion cells. The absence of Dp427 does not cause a severe phenotype but alters the neuronal architecture, compromising mainly the pre-synaptic photoreceptor terminals and their post-synaptic sites. The analysis of two dystrophic mutants of the fruit fly Drosophila melanogaster, the homozygous Dys(E17) and Dys(EP3397), lacking functional large-isoforms of dystrophin-like protein, revealed rhabdomere degeneration. Structural damages were evident in the internal network of retina/lamina where photoreceptors make the first synapse. Both accumulated autophagosomes and apoptotic features were detected and the visual system was functionally impaired. The reactivation of the autophagosome turnover by rapamycin prevented neuronal cell death and structural changes of mutant flies and, of interest, sustained autophagy ameliorated their response to light. Overall, these findings indicate that functional full-length dystrophin is required for synapsis stabilization and neuronal survival of the retina, allowing also proper autophagy as a prerequisite for physiological cell fate and visual properties (Catalani, 2020).

    Drosophila NUAK functions with Starvin/BAG3 in autophagic protein turnover

    The inability to remove protein aggregates in post-mitotic cells such as muscles or neurons is a cellular hallmark of aging cells and is a key factor in the initiation and progression of protein misfolding diseases. While protein aggregate disorders share common features, the molecular level events that culminate in abnormal protein accumulation cannot be explained by a single mechanism. This study shows that loss of the serine/threonine kinase NUAK causes cellular degeneration resulting from the incomplete clearance of protein aggregates in Drosophila larval muscles. In NUAK mutant muscles, regions that lack the myofibrillar proteins F-actin and Myosin heavy chain (MHC) instead contain damaged organelles and the accumulation of select proteins, including Filamin (Fil) (Cheerio) and CryAB. NUAK biochemically and genetically interacts with the cochaperone Starvin (Stv), the ortholog of mammalian Bcl-2-associated athanogene 3 (BAG3). Consistent with a known role for the co-chaperone BAG3 and the Heat shock cognate 71 kDa (HSC70)/HSPA8 ATPase in the autophagic clearance of proteins, RNA interference (RNAi) of Drosophila Stv, Hsc70-4, or autophagy-related 8a (Atg8a) all exhibit muscle degeneration and muscle contraction defects that phenocopy NUAK mutants. It was further demonstrated that Fil/Cheerio is a target of NUAK kinase activity and abnormally accumulates upon loss of the BAG3-Hsc70-4 complex. In addition, Ubiquitin (Ub), ref(2)p/p62, and Atg8a are increased in regions of protein aggregation, consistent with a block in autophagy upon loss of NUAK. Collectively, these results establish a novel role for NUAK with the Stv-Hsc70-4 complex in the autophagic clearance of proteins that may eventually lead to treatment options for protein aggregate diseases (Brooks, 2020).

    Proteins must fold into an intrinsic three dimensional structure to perform distinct cellular functions. Denatured or misfolded proteins can be refolded by chaperones or are subject to degradation by the ubiquitin-proteasome system (UPS) and/or the autophagosome-lysosome pathway (ALP). The accumulation of misfolded proteins upon genetic mutation or decreased chaperone function causes protein aggregates that are not effectively cleared by the UPS or the ALP. Environmental insults or aging may exacerbate this accumulation of misfolded proteins, resulting in disease and eventual cell death (Brooks, 2020).

    A specialized autophagy pathway, termed chaperone-assisted selective autophagy (CASA), has been verified in both Drosophila and mammalian systems. The CASA complex includes BAG3 in concert with the chaperones HSC70/HSPA8 (HSP70 family), HSPB8 (small HSP family), and the ubiquitin (Ub) ligase CHIP/STUB1. CASA regulates the removal and degradation of Fil from the Z-disc in striated muscle or actin stress fibers in non-muscle cells. The N-terminal actin-binding domain (ABD) in Fil is followed by multiple immunoglobulin (Ig)-like repeats which bind numerous proteins to link the internal cytoskeleton to the sarcolemma. Tension exerted by contractile muscle tissue requires continuous folding and refolding of individual Ig-like domains in Fil, eventually damaging the ability of the protein to sense and transmit mechanical strain. The BAG3-HSC70 protein complex binds to the mechanosensor region (MSR) of Fil and upon detection of protein damage, CHIP ensures the addition of polyubiquitin (polyUb) moieties. Rather than promoting delivery to the proteasome, these Ub chains instead recruit the autophagic Ub adapter protein p62/SQSTM1. p62 interacts with Atg8a/LC3 to induce autophagophore formation and the subsequent clearance of Fil through lysosomal degradation. Fil aggregates and a block in autophagosome-lysosome fusion are present in lysosomal associated membrane protein 2 (LAMP2)-deficient muscles, thus linking impaired autophagy to abnormal protein deposits (Brooks, 2020).

    Drosophila NUAK encodes for a conserved serine/threonine kinase that is homologous to the mammalian kinases NUAK1/ARK5 and NUAK2/SNARK. These proteins comprise a family of twelve AMP-activated protein kinase (AMPK)-related kinases (NUAK1 and 2, BRSK 1 and 2, QIK, QSK, SIK, MARK 1-4, and MELK) that share a conserved N-terminal kinase domain activated by the upstream liver kinase B1 (LKB1). NUAK1 and NUAK2 proteins are broadly expressed, but enriched in cardiac and skeletal muscle. Muscle contraction and LKB1 phosphorylation can activate both NUAK proteins. NUAK2 activity is additionally stimulated by oxidative stress, AMP, and glucose deprivation in various cell types. Interestingly, NUAK2 expression increases during muscle differentiation and in response to stress or in aging muscle tissue, whereas dominant-negative (DN)-NUAK2 induces atrophy. Homozygous NUAK1 KO mice are embryonic lethal and <10% of NUAK2 homozygotes survive, precluding analysis of post-embryonic contributions. Because of this embryonic lethality, conditional NUAK1 KO mice were generated to examine muscle function . However, no change was observed in muscle mass or fiber size between control or muscle-specific NUAK1 KO mice, likely due to functional redundancy (Brooks, 2020).

    The presence of single NUAK orthologs in worms (Unc-82) or flies (NUAK/CG43143) allows for the study of NUAK protein function without compensation from additional family members that may mask cellular roles. unc-82 associates with Paramyosin and likely Myosin B to promote proper myofilament assembly in C. elegans. The kinase domain in Drosophila NUAK shares 61% identity and 80% similarity to human NUAK1 and NUAK2. In flies, RNAi knockdown of NUAK phenocopies weak Lkb1 defects in regulating cell polarity during ommatidial formation and actin cone formation in spermatogenesis. NUAK kinase targets or additional functions in other tissues have not been reported (Brooks, 2020).

    This study identified Drosophila NUAK as a key regulator of autophagic protein clearance in muscle tissue. NUAK physically interacts with and phosphorylates Fil [encoded by Drosophila cheerio (cher)]. NUAK also genetically and biochemically interacts with the Stv-Hsc-70-4 complex and Stv overexpression is sufficient to rescue NUAK-mediated muscle deterioration. The identification of Fil as a cargo protein that abnormally accumulates in muscle tissue deficient for NUAK, Stv, Hsc70-4, and Atg8a links protein aggregation to defects in autophagic disposal (Brooks, 2020).

    Prior to this study, few substrates of NUAK kinase activity had been uncovered. One of these is Myosin phosphatase targeting-1 (MYPT1), a regulatory subunit of myosin light-chain phosphatase. Two Drosophila regulatory subunits, MYPT75D and Myosin binding subunit (Mbs), were tested in Stv NUAK sensitized genetic assay and no protein aggregation and/or muscle degeneration was observed. While negative, this data nevertheless argues that this family of phosphatases likely does not function with NUAK in muscle tissue. Since the mammalian NUAK1-MYPT1 interaction was identified in vitro and further validated in HEK293 cells, NUAK likely has cell and tissue-specific targets that regulate diverse biological outputs (Brooks, 2020).

    Based upon the discovery of Fil as a novel NUAK substrate, two scenarios are envisioned that are not mutually exclusive to explain the molecular function of NUAK in preventing protein aggregation. First, the increase in sarcomere number upon muscle-specific NUAK RNAi suggests that at least one role of NUAK may be to negatively regulate the addition of proteins (such as Fil) into sarcomeres. This data is consistent with studies that show C. elegans unc-82 regulates myofilament assembly. Notably, one key feature of the misincorporated proteins in unc-82 mutants is their inclusion into aggregate-like structures, similar to the accumulation of Fil and CryAB in NUAK-/- muscles. An additional, or alternative possibility, is that NUAK phosphorylates unfolded or 'damaged' Fil for removal from the sarcomere, thereby triggering the Stv-Hsc70-4 complex to promote autophagic turnover. Thus, proteins such as Fil that fail to get incorporated into sarcomeres and/or sustain damage due to repeated rounds of tension-induced muscle contraction, may destabilize myofilament architecture and trigger abnormal protein (Brooks, 2020).

    In both contractile muscle tissue and in adherent cells subjected to mechanical force, BAG3 acts as a hub to coordinate Fil-induced tension-sensing and autophagosome formation. The MSR of Fil is comprised of Ig repeats whose conformational transitions between open and closed states dictate differential protein-protein interactions and biological outputs. While the chaperones Hsc70/HSPA8 and HSPB8 weakly bind to the MSR of Fil, this biochemical interaction is greatly enhanced in the presence of BAG3. Interestingly, BAG3 interacts with Ig repeats 19-21 in the MSR, while the selected interaction domain of NUAK with Fil comprises Ig repeats 15-18. These data suggest that NUAK and Stv each bind to a separate region of the MSR in Fil (Brooks, 2020).

    It remains to be determined if NUAK-mediated phosphorylation is a prerequisite for the removal of damaged Fil protein by BAG3. The rescue results suggest that this phosphorylation event is not required as Stv overexpression alleviates protein aggregation and muscle degeneration upon a loss of NUAK. An alternative possibility is that this excess Stv protein is present in sufficient amounts to interact with Fil and overcome the necessity for phosphorylation by NUAK. The inability of NUAK overexpression to restore muscle defects due to knockdown of Stv, Hsc70-4, or Atg8a suggests that NUAK functions upstream or parallel to this pathway. It seems likely that NUAK has additional target substrates for kinase activity that may regulate autophagic protein clearance in muscle tissue (Brooks, 2020).

    Recent studies demonstrate that increased autophagic degradation of Fil by BAG3 also induces fil transcription as a compensatory mechanism to ensure steady-state Fil levels. Thus, whether loss of NUAK or Stv alters gene expression upon a block in protein clearance was investigated. While the mRNA levels of cher, CryAB, Hsc70-4, or Atg8a were not altered in NUAK or stv mutants, there was a large increase in p62 transcripts. Thus, this increase in p62 mRNA synthesis may contribute to the elevated p62 protein levels observed upon loss of NUAK or Stv as multiple stress conditions increase p62 transcription, including proteasome inhibition, starvation and atrophic muscle conditions. Data that support a role for an autophagic block include the localization of p62 and Atg8a to regions of protein aggregation (Brooks, 2020).

    A model for NUAK is proposed that incorporates these new findings with existing roles for BAG3. Fil and CryAB are physically associated at the Z-disc in Drosophila larval muscle. The phosphorylation of Fil by NUAK may control the incorporation of Fil into the Z-disc during myofibril assembly and/or may be required for the disposal of damaged Fil protein. BAG3 and chaperones such as Hsc70/HSPA8 are thought to monitor the MSR of Fil to detect force-induced damage and to promote the addition of K63-linked polyUb chains. Recruitment of the ubiquitin autophagic adapter p62/SQSTM1 induces autophagosome initiation through the accumulation of Atg8a. Eventual fusion of these autophagosomes with lysosomes promotes protein client complex destruction (Brooks, 2020).

    Upon loss of NUAK, excess Fil protein that fails to be incorporated into the Z-disc and/or is damaged due to tension-induced muscle contraction begins to accumulate near the Z-disc. The presence of CryAB in Fil-like aggregates may be due to the normal association of CryAB with Fil at the Z-disc, either to monitor Fil protein damage, or to prevent protein aggregation. It is interesting that while both Fil and CryAB contain actin-binding domains, these associations are lost in NUAK-/- muscle tissue as F-actin is displaced from regions of Fil-CryAB accumulation. At this point it cannot be determined if NUAK preferentially binds to the short (~90kD) and/or long (~240 kD) Fil isoforms since the mapped interaction domains (Ig domains 15-18) are present in both isoforms (Brooks, 2020).

    In the initial stages of aggregate formation, nearly all Fil puncta are decorated with Ub. It is hypothesized that the observed decrease in Ub-Fil colocalization in large regions of aggregate formation may be due to intrinsic properties of aggregation-prone proteins whereby protein misfolding triggers aggregation of Fil with itself and other proteins. The accumulation of p62 and circular structures that stain positive for Atg8a in regions of Fil accumulation demonstrate that the autophagosome machinery is recruited to BAG3-client complexes. The absence of lysosomes in these aggregate regions suggest that either fusion and/or transport to sites of degradation are compromised (Brooks, 2020).

    CASA-mediated autophagy via the BAG3-client complex includes Hsc70-4/HSPA8, HSPB8, and the E3 ligase CHIP/STUB1, the latter of which ubiquitinates Fil for the subsequent recruitment of p62 to initiate autophagosome formation. However, fibroblasts deficient for CHIP are not defective in autophagy and mice or flies lacking CHIP/STUB1 are viable. A failure to enhance protein aggregation defects upon CHIP RNAi knockdown in the sensitized NUAK+/- or stv+/- backgrounds suggests that additional Ub ligases cooperate with the Stv/BAG3 complex to remove damaged proteins. Future studies will also determine which Drosophila protein is the equivalent of HSPB8 since no genetic interactions were observed with putative CG14207 or Hsp67Bc RNAi lines. This negative data does not rule out the possibility that protein levels are not reduced enough to see phenotypes upon RNAi induction or possible functional redundancy exists between CG14207 and Hsp67Bc (Brooks, 2020).

    An interesting hallmark of protein aggregate diseases is the accumulation of specific proteins in affected cells or tissues. Thus, proteins susceptible to aggregation in vivo may possess specific structural characteristics or shared biological functions. This latter feature is evident in a group of protein aggregate diseases termed myofibrillar myopathies (MFM). Laser microdissection of aggregates from normal or affected muscles reveal specificity in the types of proteins that accumulate in patients afflicted with MFMs. Common proteins present in these aggregates include Filamin C (FILC), αB-crystallin (CRYAB), BAG3, and Desmin (DES), among others. The inability of MFM patients to clear these aggregates results in myofibrillar degeneration and a decline in muscle function. Interestingly, mutations in Drosophila NUAK phenocopy both structural and functional deficits observed in MFM patients, including Fil and CryAB accumulation, muscle degeneration, and locomotor defects. The discovery of cellular degeneration and protein aggregation in muscle tissue upon loss of the single fly NUAK ortholog highlights the power of Drosophila as a model. Future studies will focus on identifying kinase targets of NUAK and defining additional proteins that function in NUAK and stv-mediated autophagy for the eventual development of therapeutic targets to treat MFMs and other protein aggregate diseases (Brooks, 2020).

    Degradation of arouser by endosomal microautophagy is essential for adaptation to starvation in Drosophila

    Hunger drives food-seeking behaviour and controls adaptation of organisms to nutrient availability and energy stores. Lipids constitute an essential source of energy in the cell that can be mobilised during fasting by autophagy. Selective degradation of proteins by autophagy is made possible essentially by the presence of LIR and KFERQ-like motifs. Using in silico screening of Drosophila proteins that contain KFERQ-like motifs, the adaptor protein Arouser, which functions to regulate fat storage and mobilisation and is essential during periods of food deprivation, was identified and characterized. Hypomorphic arouser mutants are not satiated, are more sensitive to food deprivation, and are more aggressive, suggesting an essential role for Arouser in the coordination of metabolism and food-related behaviour. This analysis shows that Arouser functions in the fat body through nutrient-related signalling pathways and is degraded by endosomal microautophagy. Arouser degradation occurs during feeding conditions, whereas its stabilisation during non-feeding periods is essential for resistance to starvation and survival. In summary, these data describe a novel role for endosomal microautophagy in energy homeostasis, by the degradation of the signalling regulatory protein Arouser (Jacomin, 2021).

    An autophagy-dependent tubular lysosomal network synchronizes degradative activity required for muscle remodeling

    Lysosomes are compartments for the degradation of both endocytic and autophagic cargoes. The shape of lysosomes changes with cellular degradative demands; however, there is limited knowledge about the mechanisms or significance that underlies distinct lysosomal morphologies. This study found an extensive tubular autolysosomal network in Drosophila abdominal muscle remodeling during metamorphosis. The tubular network transiently appeared and exhibited the capacity to degrade autophagic cargoes. The tubular autolysosomal network was uniquely marked by the autophagic SNARE protein Syntaxin17 and its formation depended on both autophagic flux and degradative function, with the exception of the Atg12 and Atg8 ubiquitin-like conjugation systems. Among ATG-deficient mutants, the efficiency of lysosomal tubulation correlated with the phenotypic severity in muscle remodeling. The lumen of the tubular network was continuous and homogeneous across a broad region of the remodeling muscle. Altogether, this study revealed that the dynamic expansion of a tubular autolysosomal network synchronizes the abundant degradative activity required for developmentally regulated muscle remodeling (Murakawa, 2020).

    EGFR-dependent suppression of synaptic autophagy is required for neuronal circuit development

    The development of neuronal connectivity requires stabilization of dynamic axonal branches at sites of synapse formation. Models that explain how axonal branching is coupled to synaptogenesis postulate molecular regulators acting in a spatiotemporally restricted fashion to ensure branching toward future synaptic partners while also stabilizing the emerging synaptic contacts between such partners. This question was investigated using neuronal circuit development in the Drosophila brain as a model system. Epidermal growth factor receptor (EGFR) activity was shown to be required in presynaptic axonal branches during two distinct temporal intervals to regulate circuit wiring in the developing Drosophila visual system. EGFR is required early to regulate primary axonal branching. EGFR activity is then independently required at a later stage to prevent degradation of the synaptic active zone protein Bruchpilot (Brp). Inactivation of EGFR results in a local increase of autophagy in presynaptic branches and the translocation of active zone proteins into autophagic vesicles. The protection of synaptic material during this later interval of wiring ensures the stabilization of terminal branches, circuit connectivity, and appropriate visual behavior. Phenotypes of EGFR inactivation can be rescued by increasing Brp levels or downregulating autophagy. In summary, a temporally restricted molecular mechanism required for coupling axonal branching and synaptic stabilization was demonstrated that contributes to the emergence of neuronal wiring specificity (Dutta, 2023).

    Lysosomal cystine mobilization shapes the response of TORC1 and tissue growth to fasting

    Adaptation to nutrient scarcity involves an orchestrated response of metabolic and signaling pathways to maintain homeostasis. This study found that in the fat body of fasting Drosophila, lysosomal export of cystine coordinates remobilization of internal nutrient stores with reactivation of the growth regulator target of rapamycin complex 1 (TORC1). Mechanistically, cystine was reduced to cysteine and metabolized to acetyl-coenzyme A (acetyl-CoA) by promoting CoA metabolism. In turn, acetyl-CoA retained carbons from alternative amino acids in the form of tricarboxylic acid cycle intermediates and restricted the availability of building blocks required for growth. This process limited TORC1 reactivation to maintain autophagy and allowed animals to cope with starvation periods. It is proposed that cysteine metabolism mediates a communication between lysosomes and mitochondria, highlighting how changes in diet divert the fate of an amino acid into a growth suppressive program (Parkhitko, 2022).

    Maintaining cellular homeostasis upon nutrient shortage is an important challenge for all animals. Decreased activity of TORC1 is necessary to limit translation, reduce growth rates, and promote autophagy. Conversely, minimal TORC1 activity is required to promote lysosomal biogenesis, thus maintaining autophagic degradation necessary for survival. Using Drosophila as an in vivo model, this study found that TORC1 reactivation upon fasting integrates the biosynthesis of amino acids from anaplerotic inputs into the control of growth. The regulation of aspartate abundance appears to be critical during this process, possibly because it serves as a cataplerotic precursor for various macromolecules, including other amino acids and nucleotides, which in turn impinge on TORC1 activity. Cysteine recycling through the lysosome may fuel acetyl-CoA synthesis and prevent reactivation of TORC1 above a threshold that would compromise autophagy and survival during fasting. Reactivation of TORC1 during fasting was not passively controlled by the extent of amino acid remobilized from the lysosome. Instead, cysteine metabolism supported an increased incorporation of the carbons from these remobilized amino acids into the TCA cycle. It is therefore proposed that the remobilized amino acids may be transiently stored in the form of TCA cycle intermediates compartmentalized in the mitochondria, thereby restricting their accessibility. The regulation of TORC1 activity over a fasting period appears to be a combination of activating and suppressing cues that conciliate autophagy with anabolism. This process is self-regulated by autophagy, because autophagic protein degradation controls cystine availability through the lysosomal cystinosin transporter. Thus, in contrast to fed conditions, in which amino acid transporters at the plasma membrane maintain high cytosolic concentration of leucine and arginine that can directly be sensed by members of the TORC1 machinery, TORC1 reactivation in prolonged fasting is regulated indirectly by lysosome-mitochondrial cross-talk. Because cystinosin has also been shown to physically interact with several components of lysosomal TORC1 in mammalian cells, additional layers of regulation are conceivable during this process (Parkhitko, 2022).

    Multiple functions of cysteine impinge on cellular metabolism, including transfer RNA thiolation, the generation of hydrogen sulfide, the regulation of hypoxia-inducible factor (HIF), and its antioxidant function through glutathione synthesis. Supplementation with cysteine or modified molecules such as N-acetyl-cysteine (NAC) can be used to efficiently buffer oxidative stress and perhaps alleviate symptoms of diseases that promote oxidative stress or glutathione deficiency, including cystinosis. Cysteine or NAC treatment extends the life span in flies, worms, and mice, and mice fed NAC show a sudden drop in body weight similar to that caused by dietary restriction. The results indicate that cysteine may not only act through its antioxidant function but also by restricting the availability of particular amino acids and limiting mTOR activity, processes known to extend life span. Moreover, this study shows that CoA is a main fate of cysteine that affects oxidative metabolism in the mitochondria, which is the main source of reactive oxygen species (ROS). Thus, the antioxidant function of cysteine also might be coupled to its effects on the mitochondria to buffer ROS production (Parkhitko, 2022).

    In summary, this study demonstrate that cysteine metabolism acts in a feedback loop involving de novo CoA synthesis, the TCA cycle, and amino acid metabolism to limit TORC1 reactivation upon prolonged fasting. This pathway may be particularly important for developing organisms that must maintain autophagy and balance growth and survival during periods of food shortage (Parkhitko, 2022).

    Bub1 and Bub3 regulate metabolic adaptation via macrolipophagy in Drosophila

    Lipophagy, the process of selective catabolism of lipid droplets (LDs) by autophagy, maintains lipid homeostasis and provides cellular energy under metabolic adaptation, yet its underlying mechanism remains largely ambiguous. This study shows that the Bub1-Bub3 complex, the crucial regulator involved in the whole process of chromosome alignment and separation during mitosis, controls the fasting-induced lipid catabolism in the fat body (FB) of Drosophila. Bidirectional deviations of the Bub1 or Bub3 level affect the consumption of triacylglycerol (TAG) of fat bodies and the survival rate of adult flies under starving. Moreover, Bub1 and Bub3 work together to attenuate lipid degradation via macrolipophagy upon fasting. Thus, this study uncovered physiological roles of the Bub1-Bub3 complex on metabolic adaptation and lipid metabolism beyond their canonical mitotic functions, providing insights into the in vivo functions and molecular mechanisms of macrolipophagy during nutrient deprivation (Zhang, 2023).

    ESCRT dysfunction compromises endoplasmic reticulum maturation and autophagosome biogenesis in Drosophila

    Autophagy targets cytoplasmic materials for degradation and influences cell health. Organelle contact and trafficking systems provide membranes for autophagosome formation, but how different membrane systems are selected for use during autophagy remains unclear. This study reports a novel function of the endosomal sorting complex required for transport (ESCRT) in the regulation of endoplasmic reticulum (ER) coat protein complex II (COPII) vesicle formation that influences autophagy. The ESCRT functions in a pathway upstream of Vps13D to influence COPII vesicle transport, ER-Golgi intermediate compartment (ERGIC) assembly, and autophagosome formation. Atg9 functions downstream of the ESCRT to facilitate ERGIC and autophagosome formation. Interestingly, cells lacking either ESCRT or Vps13D functions exhibit dilated ER structures that are similar to cranio-lenticulo-sutural dysplasia patient cells with SEC23A mutations, which encodes a component of COPII vesicles. These data reveal a novel ESCRT-dependent pathway that influences the ERGIC and autophagosome formation (Wang, 2022).

    The ESCRT regulates autophagy in multiple ways, including AP membrane sealing and autolysosome (ALY) formation. This study reveals a previously undescribed function of the ESCRT in ER trafficking and autophagy. The findings indicate that the ESCRT is required for COPII vesicle-derived compartments and AP formation. Loss of ESCRT components influence COPII vesicle marker formation and ER morphology. This study also shows that Vps13D co-localizes with markers of different stages of COPII vesicle maturation between the ER and Golgi apparatus, including markers of ERGIC formation. In the absence of ESCRT and Vps13D function, ER structure is altered in a manner that is similar to CLSD patient cells with SEC23A mutations. In addition, the ESCRT impacts Atg9 distribution. Vps13D and Atg9 are required for autophagy and appear to influence the ERGIC for the induction of autophagy. Therefore, this study identifies previously unrecognized steps in a regulatory pathway for autophagy, including novel roles for the ESCRT and Vps13D in COPII-derived and ERGIC-associated AP formation (Wang, 2022).

    Autophagy involves a core mechanism, but some autophagy regulatory mechanisms vary in different cell contexts in animals. During Drosophila midgut development, most of the core autophagy genes are required, but Atg7 and Atg3 are not required for cell size reduction, protein, and organelle clearance. Instead, the ubiquitin activating enzyme Uba1 and ubiquitin function in autophagy. Vps13D contains a conserved ubiquitin-binding UBA domain and is required for developmental autophagy in the fly intestine. Thus, Vps13D has properties of an autophagy receptor and therefore may link ubiquitin, autophagy proteins, and their autophagic cargo substrates. Alternatively, ubiquitin may have cargo-receptor-independent functions in autophagy during intestine development (Wang, 2022).

    Tsg101 and Vps36 both possess ubiquitin-binding domains and are required for autophagy in the fly intestine. While previous studies have shown that the ESCRT functions in AP membrane sealing and ALY formation, studies of Tsg101, Vps36, and all other ESCRT components tested indicate a novel role in ER trafficking for AP formation in the intestine. This study systematically compared ESCRT function in different cell contexts in Drosophila. Consistent with previous studies, ESCRT mutant fat body cells accumulate Atg8a puncta indicating that autophagy is blocked following ALY formation. By contrast, the ESCRT is required for Atg8a puncta formation in intestine cells. Therefore, the ESCRT machinery functions in a cell context-specific manner to regulate autophagy (Wang, 2022).

    ESCRT machinery regulates multiple dynamic membrane changes, including vesicle abscission, membrane sealing, and repair. The function of the ESCRT in the COPII pathway is novel but also has similarities to the essential function of the ESCRT in membrane remodeling. During Drosophila development, Hrs is distributed near ER and co-localized with Ergic53 when autophagy is induced. In ESCRT mutant cells, both COPII and Ergic53 puncta were decreased and dissociated, indicating that COPII vesicles derived from ER are inhibited without the ESCRT. Multiple membrane remodeling processes require the ESCRT, including vesicle budding and internalization. Thus, it appears that the ESCRT affects COPII-coated ER exit sites to shed vesicles and influences ERGIC and AP assembly in a cell-specific manner. Decreased function of ESCRT regulatory genes influences Vps13D levels. By contrast, Vps13D regulates COPII regulatory protein localization, suggesting that Vps13D functions downstream of the ESCRT to influence COPII. It should be noted, however, that it is difficult to exclude the possibility that the ESCRT may also participate in either AP membrane sealing or fusion between AP and lysosome at a later stage in the autophagy process. In addition, we cannot exclude a possible indirect influence of the ESCRT on the ER and COPII maturation that is independent of Vps34 (Wang, 2022).

    The roles of the regulators of COPII vesicle and ERGIC formation in autophagy may be Drosophila intestine cell context specific. However, COPII and the ERGIC have been implicated in the regulation of autophagy in both yeast and mammalian cells. Atg9 interacts with membrane compartments from the secretory pathway, endocytic vesicles, and Golgi apparatus in both yeast and mammals. Atg9 interacts with the COPII component Sec24 in yeast, and co-fractionates with ERGIC components in mammalian cells. Thus, it is likely that membranes from COPII compartments are utilized for autophagy induction. Atg9 is conserved in Drosophila, and the current analyses indicate that Atg9 is downstream of the ESCRT, Vps13D, and the ERGIC. Therefore, the ERGIC is also a potential membrane source for Atg9 to regulate the induction of autophagy in Drosophila, consistent with ERGIC function in mammalian cells. Thus, it is possible that the roles of COPII, Vps13D, and the ERGIC are more prevalent in the regulation of autophagy than previously recognized. Future studies should reveal how broadly these factors function in autophagy, as well as the role of the ESCRT in COPII vesicle maturation in other cell types (Wang, 2022).

    TFEB/Mitf links impaired nuclear import to autophagolysosomal dysfunction in C9-ALS

    Disrupted nucleocytoplasmic transport (NCT) has been implicated in neurodegenerative disease pathogenesis; however, the mechanisms by which disrupted NCT causes neurodegeneration remain unclear. A Drosophila screen identified ref(2)P/p62, a key regulator of autophagy, as a potent suppressor of neurodegeneration caused by the GGGGCC hexanucleotide repeat expansion (G4C2 HRE) in C9orf72 that causes amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). p62 is increased and forms ubiquitinated aggregates due to decreased autophagic cargo degradation. Immunofluorescence and electron microscopy of Drosophila tissues demonstrate an accumulation of lysosome-like organelles that precedes neurodegeneration. These phenotypes are partially caused by cytoplasmic mislocalization of Mitf/TFEB, a key transcriptional regulator of autophagolysosomal function. Additionally, TFEB is mislocalized and downregulated in human cells expressing GGGGCC repeats and in C9-ALS patient motor cortex. These data suggest that the C9orf72-HRE impairs Mitf/TFEB nuclear import, thereby disrupting autophagy and exacerbating proteostasis defects in C9-ALS/FTD (Cunningham, 2020).

    Lipid profiles of autophagic structures isolated from wild type and Atg2 mutant Drosophila

    Autophagy is mediated by membrane-bound organelles and it is an intrinsic catabolic and recycling process of the cell, which is very important for the health of organisms. The biogenesis of autophagic membranes is still incompletely understood. In vitro studies suggest that Atg2 protein transports lipids presumably from the ER to the expanding autophagic structures. Autophagy research has focused heavily on proteins and very little is known about the lipid composition of autophagic membranes. This study describes a method for immunopurification of autophagic structures from Drosophila melanogaster (an excellent model to study autophagy in a complete organism) for subsequent lipidomic analysis. Western blots of several organelle markers indicate the high purity of the isolated autophagic vesicles, visualized by various microscopy techniques. Mass spectrometry results show that phosphatidylethanolamine (PE) is the dominant lipid class in wild type (control) membranes. In Atg2 mutants (Atg2-), phosphatidylinositol (PI), negatively charged phosphatidylserine (PS), and phosphatidic acid (PA) with longer fatty acyl chains accumulate on stalled, negatively charged phagophores. Tandem mass spectrometry analysis of lipid species composing the lipid classes reveal the enrichment of unsaturated PE and phosphatidylcholine (PC) in controls versus PI, PS and PA species in Atg2-. Significant differences in the lipid profiles of control and Atg2- flies suggest that the lipid composition of autophagic membranes dynamically changes during their maturation. These lipidomic results also point to the in vivo lipid transport function of the Atg2 protein, pointing to its specific role in the transport of short fatty acyl chain PE species (Laczko-Dobos, 2021).

    Macroautophagy (autophagy hereafter) is a 'self-eating' process of eukaryotic cells, during which damaged or obsolete cytoplasmic components (organelles, proteins, lipids, sugars etc.) are removed and degraded in lysosomes\. Misregulation of autophagy contributes to neurodegenarative diseases, cancer, accelerated aging, etc. This fundamental catabolic process relies on the biogenesis of unique, very dynamic membranes and membrane-bound autophagic vesicles. The pathway initiates with the nucleation of a double-membrane structure called phagophore (formerly also known as isolation membrane), which expands and engulfs a portion of the cytoplasm. After closure and sealing, it will form the autophagosome, a double membrane vesicle. In the last step, autophagosomes will fuse with lysosomes to form autolysosomes, where the sequestered cytoplasmic material will be degraded and recycled back to the cytosol (Laczko-Dobos, 2021).

    Fluorescence and electron microscopy are widely used to study these specific organelles. Autophagic structures can be isolated for example from human cell lines, yeast, mouse and rat tissues by applying subcellular fractionation, immunoprecipitation or combination of these two methods. Fruit flies are an excellent model organism to study autophagy in a complete animal as they can be genetically manipulated very easily, and about 75% of their genes show homology with disease associated human genes, so they can serve as models for various human diseases (Laczko-Dobos, 2021).

    Autophagosomes are unique organelles regarding their lipid and protein composition, morphology and biogenesis. Autophagosome formation occurs very rapidly upon induction of autophagy, which requires a tremendous amount of membrane source(s). De novo synthesis of autophagic membranes is the most enigmatic field of autophagy; almost every compartment of the endomembrane system has been implicated in this process. Interestingly, also the contact sites between organelles such as ER and mitochondria may play an important role in this biogenesis event. During phagophore expansion, the synthesis or delivery of lipids must be distinctly controlled. Formation of these specific membranes relies on a collaborative work between proteins encoded by autophagy-related (Atg) genes and membrane lipids (Laczko-Dobos, 2021).

    It has been shown recently that Atg2 protein is not only a potential tether between ER and phagophores, but it may also transport lipids from the ER to promote autophagosome biogenesis (Osawa, 2019a; Osawa, 2019b). Several in vitro studies on yeast (and human) Atg2 (Atg2A and B) showed the lipid transport activity of this special protein, which is able to transport several lipids at once using its hydrophobic cavity (Osawa, 2019b; Valverde, 2019; Maeda, 2019; Osawa, 2020). A very recent discovery is that the fast expansion of autophagic membranes after autophagy induction relies on localized 'on-demand' de novo phospholipid synthesis, by the aid of Acyl-CoA synthetase Faa1 enzymes identified on nucleated phagophores in yeast, and the Atg2-Atg18 protein complex may also be involved in this process. The P-element induced Drosophila mutant (Atg2EP3697), bearing the transposon insertion at the 5' non-translated region of the Atg2 gene that resulted in a strong hypomorphic allele, showed an autophagy defect similar to mammalian and C. elegans Atg2 mutants [26,27]. Microscopy and biochemical investigations support that loss of Atg2 protein function in Drosophila, worms, and also in mammalian cells causes a sealing defect of phagophores, leading to accumulation of enlarged phagophore-like structures. Other key players of autophagy are Atg8 proteins together with their lipid conjugation (including the E3-like Atg5-Atg12-Atg16 complex) and deconjugation machinery. They have important roles in the biogenesis of autophagic membranes, and they are reversebly conjugated to the PE head groups present in the membranes of autophagic structures. The two distinct forms of Atg8 proteins: the non-lipidated Atg8 (Atg8-I) and lipidated Atg8 (Atg8-II) are the most widely used autophagic markers, including Atg8a in Drosophila (Laczko-Dobos, 2021).

    Lipids are largely unexplored players of the phagophore biogenesis machinery. The major structural lipids in eukaryotic membranes are glycerophospholipids, sphingolipids and sterols. The most abundant glycerophospholipid classes of Drosophila are: phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PI) and less abundant lipid classes are phosphatidylglycerol (PG) and phosphatidic acid (PA). PC accounts for more than 50% of the phospholipids in most eukaryotic membranes. Interestingly, in Drosophila, PE is the most abundant structural lipid and also a component of the lipoproteins (like Atg8), in contrast with more PC-centric lipidome of mammalian cells. These main lipid classes differ in the chemical composition of their head groups and in length and saturation level of their fatty acyl chains. This determines the shape and physicochemical behavior of these lipid molecules, such as phase transition properties and thickness of the membranes. All these properties influence membrane curvature and fusion events (Laczko-Dobos, 2021).

    Lipids are not only structural components of the membranes but they also play a regulatory role in cellular processes, such as autophagy. Inactivation of Desat1 (desaturase coding gene, responsible for double bond generation) in Drosophila resulted in an autophagic defect: autolysosomes could not form properly. Glycerolipids play important roles in the initiation of autophagy, elongation of phagophores, autophagosome maturation as well as in autophagosome-lysosome fusion. Different phosphorylated forms of PI including PI3P, PI4P, PI(4,5)P2, PI(3,5)P2 are also found in autophagic membranes, as different types of kinases present at these membranes are responsible for their generation. Although they are minor lipids (representing less than 1% of total lipids), together with PE they play important roles in autophagosome biogenesis by influencing the recruitment of specific proteins to the membrane. Interestingly, phosphatidic acid (PA) molecules may directly affect the physicochemical properties of lipid bilayers independently of protein effectors (Laczko-Dobos, 2021).

    This study established a method for isolating autophagic structures from adult Drosophila melanogaster, which was optimized for subsequent lipidomic investigations. Deciphering the lipid composition of autophagic membranes is crucial to fully understand the mechanism of autophagy. Using powerful Drosophila genetics and the advantage of high-throughput mass spectrometry, this study points to the important in vivo lipid transport function of Atg2 protein (Laczko-Dobos, 2021).

    Yorkie drives supercompetition by non-autonomous induction of autophagy via bantam microRNA in Drosophila

    Mutations in the tumor-suppressor Hippo pathway lead to activation of the transcriptional coactivator Yorkie (Yki), which enhances cell proliferation autonomously and causes cell death non-autonomously. The mechanism by which Yki causes cell death in nearby wild-type cells, a phenomenon called supercompetition, and its role in tumorigenesis remained unknown. This study shows that Yki-induced supercompetition is essential for tumorigenesis and is driven by non-autonomous induction of autophagy. Clones of cells mutant for a Hippo pathway component fat activate Yki and cause autonomous tumorigenesis and non-autonomous cell death in Drosophila eye-antennal discs. This study found that mutations in autophagy-related genes or NF-κB genes in surrounding wild-type cells block both fat-induced tumorigenesis and supercompetition. Mechanistically, fat mutant cells upregulate Yki-target microRNA bantam, which elevates protein synthesis levels via activation of TOR signaling. This induces elevation of autophagy in neighboring wild-type cells, which leads to downregulation of IκB Cactus and thus causes NF-κB-mediated induction of the cell death gene hid. Crucially, upregulation of bantam is sufficient to make cells to be supercompetitors and downregulation of endogenous bantam is sufficient for cells to become losers of cell competition. These data indicate that cells with elevated Yki-bantam signaling cause tumorigenesis by non-autonomous induction of autophagy that kills neighboring wild-type cells (Nagata, 2022).

    The data reveal that the Hippo pathway mutant fat clones cause supercompetition by inducing autophagy-mediated cell death in surrounding wild-type cells via NF-κB-mediated induction of hid. The autophagy induction in wild-type cells depends on Yki-bantam-mediated activation of TOR signaling in neighboring fat mutant cells. This mechanism is similar to what was observed in the elimination of ribosomal protein or Hel25E mutant loser clones when surrounded by wild-type cells. This is particularly interesting in two ways: first, it suggests that different types of cell competition, namely elimination of unfit cells by wild-type cells and elimination of wild-type cells by supercompetitors, are driven by the common mechanism, and second, it indicates that induction of autophagy in loser cells is non-autonomous, as even wild-type cells elevate autophagy when juxtaposed to supercompetitors. Although the mechanism by which autophagy is induced in loser cells nearby winner cells remains unknown, observations in this study in conjunction with the previous data on the elimination of ribosomal protein or Hel25E mutant clones suggest the possibility that relative difference in protein synthesis levels between cells plays a critical role in autophagy induction (Nagata, 2022).

    The mechanism by which elevated autophagy induces hid expression via NF-κB still remains to be elucidated. Elevated autophagy results in downregulation of IκB protein Cactus. IκB is known to be degraded by the ubiquitin-proteasome system. On the other hand, elevated autophagy by starvation or rapamycin treatment was shown to cause degradation of IκB and thus activate NF-κB in mouse fibroblast. Together, the data suggest the possibility that IκB is degraded by selective autophagy in losers of cell competition (Nagata, 2022).

    The observations of this studsy intriguingly show that non-autonomous cell death in wild-type cells promotes fat-induced tumorigenesis. This supports the idea that cancer cells expand their territories within the tissue by cell competition during malignant progression of tumors. While the mechanism by which wild-type cell death fuels neighboring tumorigenesis is an important open question, it may involve compensatory proliferation triggered by mitogenic factors secreted from dying cells. Intriguingly, it has been reported in Drosophila eye-antennal discs that clones of malignant tumors caused by Ras activation and cell polarity defects induce autophagy in surrounding wild-type cells, which in this case do not cause cell death but provide nutrient such as amino acids to neighboring tumors to promote their growth. Clones of cells overexpressing activated form of Yki were also shown to induce autophagy in neighboring cells, but in this case non-autonomous autophagy does not have a role in promoting tumorigenesis. Thus, non-autonomous autophagy may have multiple roles and mechanisms in regulating tissue homeostasis and tumorigenesis (Nagata, 2022).

    Given that the Hippo pathway is conserved throughout evolution and that YAP-mediated cell competition occurs in mammalian systems as well, autophagy-mediated cell death may play an important role in mammalian cell competition. Notably, in a mouse liver cancer model, hyperactivation of YAP in peritumoral hepatocytes triggers regression of primary liver tumors and melanoma-derived liver metastases. Thus, further studies on the mechanism of Hippo-signaling-mediated supercompetition in Drosophila may provide a novel therapeutic strategy against human cancers (Nagata, 2022).

    V-ATPase controls tumor growth and autophagy in a Drosophila model of gliomagenesis

    Glioblastoma (GBM), a very aggressive and incurable tumor, often results from constitutive activation of EGFR (epidermal growth factor receptor) and of phosphoinositide 3-kinase (PI3K). To understand the role of autophagy in the pathogenesis of glial tumors in vivo, an established Drosophila melanogaster model of glioma was used based on overexpression in larval glial cells of an active human EGFR and of the PI3K homolog Pi3K92E/Dp110. Interestingly, the resulting hyperplastic glia express high levels of key components of the lysosomal-autophagic compartment, including vacuolar-type H(+)-ATPase (V-ATPase) subunits and ref(2)P (refractory to Sigma P), the Drosophila homolog of SQSTM1/p62. However, cellular clearance of autophagic cargoes appears inhibited upstream of autophagosome formation. Remarkably, downregulation of subunits of V-ATPase, of Pdk1, or of the Tor (Target of rapamycin) complex 1 (TORC1) component raptor prevents overgrowth and normalize ref(2)P levels. In addition, downregulation of the V-ATPase subunit VhaPPA1-1 reduces Akt and Tor-dependent signaling and restores clearance. Consistent with evidence in flies, neurospheres from patients with high V-ATPase subunit expression show inhibition of autophagy. Altogether, these data suggest that autophagy is repressed during glial tumorigenesis and that V-ATPase and MTORC1 components acting at lysosomes could represent therapeutic targets against GBM (Formica, 2021).

    Proteostasis failure and mitochondrial dysfunction leads to aneuploidy-induced senescence

    Aneuploidy, an unbalanced number of chromosomes, is highly deleterious at the cellular level and leads to senescence, a stress-induced response characterized by permanent cell-cycle arrest and a well-defined associated secretory phenotype. This study used a Drosophila epithelial model to delineate the pathway that leads to the induction of senescence as a consequence of the acquisition of an aneuploid karyotype. Whereas aneuploidy induces, as a result of gene dosage imbalance, proteotoxic stress and activation of the major protein quality control mechanisms, near-saturation functioning of autophagy leads to compromised mitophagy, accumulation of dysfunctional mitochondria, and the production of radical oxygen species (ROS). This study uncovered a role of c-Jun N-terminal kinase (JNK) in driving senescence as a consequence of dysfunctional mitochondria and ROS. Activation of the major protein quality control mechanisms and mitophagy dampens the deleterious effects of aneuploidy, and this study has identified a role of senescence in proteostasis and compensatory proliferation for tissue repair (Joy, 2021).

    Circadian autophagy drives iTRF-mediated longevity

    Time-restricted feeding (TRF) has recently gained interest as a potential anti-ageing treatment for organisms from Drosophila to humans. TRF restricts food intake to specific hours of the day. Because TRF controls the timing of feeding, rather than nutrient or caloric content, TRF has been hypothesized to depend on circadian-regulated functions; the underlying molecular mechanisms of its effects remain unclear. To exploit the genetic tools and well-characterized ageing markers of Drosophila, this study developed an intermittent TRF (iTRF) dietary regimen that robustly extended fly lifespan and delayed the onset of ageing markers in the muscles and gut. iTRF enhanced circadian-regulated transcription, and iTRF-mediated lifespan extension required both circadian regulation and autophagy, a conserved longevity pathway. Night-specific induction of autophagy was both necessary and sufficient to extend lifespan on an ad libitum diet and also prevented further iTRF-mediated lifespan extension. By contrast, day-specific induction of autophagy did not extend lifespan. Thus, these results identify circadian-regulated autophagy as a critical contributor to iTRF-mediated health benefits in Drosophila. Because both circadian regulation and autophagy are highly conserved processes in human ageing, this work highlights the possibility that behavioural or pharmaceutical interventions that stimulate circadian-regulated autophagy might provide people with similar health benefits, such as delayed ageing and lifespan extension (Ulgherait, 2021).

    Pleiotropic role of Drosophila phosphoribosyl pyrophosphate synthetase in autophagy and lysosome homeostasis

    Phosphoribosyl pyrophosphate synthetase (PRPS) is a rate-limiting enzyme whose function is important for the biosynthesis of purines, pyrimidines, and pyridines. Importantly, while missense mutations of PRPS1 have been identified in neurological disorders such as Arts syndrome, how they contribute to neuropathogenesis is still unclear. This study identified the Drosophila ortholog of PRPS (dPRPS) as a direct target of RB/E2F in Drosophila, a vital cell cycle regulator, and engineered dPRPS alleles carrying patient-derived mutations. Interestingly, while they are able to develop normally, dPRPS mutant flies have a shortened lifespan and locomotive defects, common phenotypes associated with neurodegeneration. Careful analysis of the fat body revealed that patient-derived PRPS mutations result in profound defects in lipolysis, macroautophagy, and lysosome function. Significantly, evidence is shown that the nervous system of dPRPS mutant flies is affected by these defects. Overall, this study has uncovered an unexpected link between nucleotide metabolism and autophagy/lysosome function, providing a possible mechanism by which PRPS-dysfunction contributes to neurological disorders (Delos Santos, 2019).

    A conserved myotubularin-related phosphatase regulates autophagy by maintaining autophagic flux

    Macroautophagy (autophagy) targets cytoplasmic cargoes to the lysosome for degradation. Like all vesicle trafficking, autophagy relies on phosphoinositide identity, concentration, and localization to execute multiple steps in this catabolic process. This study screened for phosphoinositide phosphatases that influence autophagy in Drosophila and identified CG3530. CG3530 is homologous to the human MTMR6 subfamily of myotubularin-related 3-phosphatases, and therefore, was named dMtmr6. dMtmr6, which is required for development and viability in Drosophila, functions as a regulator of autophagic flux in multiple Drosophila cell types. The MTMR6 family member MTMR8 has a similar function in autophagy of higher animal cells. Decreased dMtmr6 and MTMR8 function results in autophagic vesicle accumulation and influences endolysosomal homeostasis (Allen, 2020).

    Cp1/cathepsin L is required for autolysosomal clearance in Drosophila

    Macroautophagy/autophagy is a highly conserved lysosomal degradative pathway important for maintaining cellular homeostasis. Much of the current knowledge of autophagy is focused on the initiation steps in this process. Recently, an understanding of later steps, particularly lysosomal fusion leading to autolysosome formation and the subsequent role of lysosomal enzymes in degradation and recycling, is becoming evident. Autophagy can function in both cell survival and cell death, however, the mechanisms that distinguish adaptive/survival autophagy from autophagy-dependent cell death remain to be established. Using proteomic analysis of Drosophila larval midguts during degradation, this study identified a group of proteins with peptidase activity, suggesting a role in autophagy-dependent cell death. Cp1/cathepsin L-deficient larval midgut cells accumulate aberrant autophagic vesicles due to a block in autophagic flux, yet later stages of midgut degradation are not compromised. The accumulation of large aberrant autolysosomes in the absence of Cp1 appears to be the consequence of decreased degradative capacity as they contain undigested cytoplasmic material, rather than a defect in autophagosome-lysosome fusion. Finally, this study found that other cathepsins may also contribute to proper autolysosomal degradation in Drosophila larval midgut cells. These findings provide evidence that cathepsins play an essential role in the autolysosome to maintain basal autophagy flux by balancing autophagosome production and turnover (Xu, 2020).

    Cell competition is driven by autophagy

    Cell competition is a quality control process that selectively eliminates unfit cells from the growing tissue via cell-cell interaction. Despite extensive mechanistic studies, the mechanism by which cell elimination is triggered has been elusive. Here, through a genetic screen in Drosophila, this study discovered that V-ATPase, an essential factor for autophagy, is required for triggering cell competition. Strikingly, autophagy is specifically elevated in prospective 'loser' cells nearby wild-type "winner" cells, and blocking autophagy in loser cells abolishes their elimination. Mechanistically, elevated autophagy upregulates a proapoptotic gene hid through NFkappaB, and the elevated hid cooperates with JNK signaling to effectively induce loser's death. Crucially, this mechanism generally applies to cell competition caused by differences in protein synthesis between cells. These findings establish a common mechanism of cell competition whereby cells with higher protein synthesis induce autophagy in their neighboring cells, leading to elimination of unfit cells (Nagata, 2019).

    The data reveal a common mechanism of cell competition whereby cells with lower protein synthesis compared to their neighbors upregulate autophagy, which induces hid through NFκB, and the elevated hid cooperates with JNK to effectively cause cell death (see Molecular mechanism of cell competition triggered by relative differences in the protein synthesis). This phenomenon was commonly observed in both eye discs and wing discs using the ey-FLP and hs-FLP-mediated mitotic clone systems. Although it has been well documented that autophagy promotes cell survival, it also causes cell death in some cellular contexts. For instance, forced activation of autophagy in Drosophila imaginal discs or amnioserosa cells causes caspase-dependent cell death. Autophagy also drives programed cell death in salivary gland and midgut during Drosophila metamorphosis. Furthermore, similar to the current observations, JNK and autophagy were shown to cooperate to cause autophagic cell death in mammalian cells. Thus, a similar autophagy-mediated cell elimination could be involved in cell competition in mammals (Nagata, 2019).

    The data show for the first time a cellular change, autophagy activation, is specifically induced in loser cells nearby winner cells as an early event in cell competition. The mechanism by which autophagy is specifically elevated at the clone boundary is currently unknown. A possible mechanism may be that loser cells with compromised protein synthesis actively uptake extracellular protein secreted by nearby wild-type winner cells, which could upregulate autophagy. Intriguingly, similar to Myc-overexpressing super-competitors, malignant RasV12/scribble-⁄- tumor cells upregulate autophagy in their surrounding wild-type cells non-autonomously, which is required for tumor growth and invasion. Thus, the current data suggest that autophagy-mediated cell competition may also drive tumor expansion during oncogenesis (Nagata, 2019).

    It would be important to investigate the mechanism by which upregulated autophagy induces hid expression via NFκB in the future studies. Given that NFκB-mediated hid expression was shown to play a key role in executing Minute cell competition and Myc super-competition, the autophagy-mediated cell elimination machinery could also involve other regulators of Minute cell competition and Myc super-competition such as Fwe, Azot, and TRR signaling. Indeed, Hel25E-induced cell competition was significantly suppressed when Fwe, Azot, or a TRR signaling component was knocked down. On the other hand, suppression of Hel25E-induced cell competition by knockdown of Atg1, Azot, or Spätzle (Spz, a secreted ligand for TRRs), was not further enhanced by co-knockdown of two or three of them. These data suggest that autophagy, Azot, and TRR signaling components are converged to a single pathway. Further studies on the mdechanism by which these components cooperate to drive cell elimination would lead to a comprehensive understanding of cell competition (Nagata, 2019).


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    Zygotically transcribed genes

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