Illuminating intercellular autophagy: A comprehensive review of cell non-autonomous autophagy.

Macro-autophagy (autophagy hereafter) is an evolutionarily conserved cellular process that has long been recognized as an intracellular mechanism for maintaining cellular homeostasis. It involves the formation of a membraned structure called the autophagosome, which carries cargo that includes toxic protein aggregates and dysfunctional organelles to the lysosome for degradation and recycling. Autophagy is primarily considered and studied as a cell-autonomous mechanism. However, recent studies have illuminated an underappreciated facet of autophagy, i.e., non-autonomously regulated autophagy. Non-autonomously regulated autophagy involves the degradation of autophagic components, including organelles, cargo, and signaling molecules, and is induced in neighboring cells by signals from primary adjacent or distant cells/tissues/organs. This review provides insight into the complex molecular mechanisms governing non-autonomously regulated autophagy, highlighting the dynamic interplay between cells within tissue/organ or distinct cell types in different tissues/organs. Emphasis is placed on modes of intercellular communication that include secreted molecules, including microRNAs, and their regulatory roles in orchestrating this phenomenon. Furthermore, we explore the multidimensional roles of non-autonomously regulated autophagy in various physiological contexts, spanning tissue development and aging, as well as its importance in diverse pathological conditions, including cancer and neurodegeneration. By studying the complexities of non-autonomously regulated autophagy, we hope to gain insights into the sophisticated intercellular dynamics within multicellular organisms, including mammals. These studies will uncover novel avenues for therapeutic intervention to modulate intercellular autophagic pathways in altered human physiology.

Atg1 modulates mitochondrial dynamics to promote germline stem cell maintenance in Drosophila.

Mitochondrial dynamics (fusion and fission) are necessary for stem cell maintenance and differentiation. However, the relationship between mitophagy, mitochondrial dynamics and stem cell exhaustion needs to be clearly understood. Here we report the multifaceted role of Atg1 in mitophagy, mitochondrial dynamics and stem cell maintenance in female germline stem cells (GSCs) in Drosophila. We found that depletion of Atg1 in GSCs leads to impaired autophagy and mitophagy as measured by reduced formation of autophagosomes, increased accumulation of p62/Ref (2)P and accumulation of damaged mitochondria. Disrupting Atg1 function led to mitochondrial fusion in developing cysts. The fusion resulted from an increase in Marf levels in both GSCs and cysts, and the fusion phenotype could be rescued by overexpression of Drp1 or by depleting Marf via RNAi in Atg1-depleted cyst cells. Interestingly, double knockdown of both Atg1:Drp1 led to the significant loss of germ cells (GCs) as compared to Atg1KD and Drp1KD. Strikingly, Atg1:Marf double knockdown leads to a dramatic loss of GSCs, GCs and a total loss of vitellogenic stages, suggesting a block in oogenesis. Overall, our results demonstrate that Drp1, Marf and Atg1 function together to influence female GSC maintenance, their differentiation into cysts and oogenesis in Drosophila.

Exploring therapeutic potential of mitophagy modulators using Drosophila models of Parkinson's disease.

Parkinson's disease (PD) is the second most popular age-associated neurodegenerative disorder after Alzheimer's disease. The degeneration of dopaminergic neurons, aggregation of α-synuclein (α-syn), and locomotor defects are the main characteristic features of PD. The main cause of a familial form of PD is associated with a mutation in genes such as SNCA, PINK1, Parkin, DJ-1, LRKK2, and others. Recent advances have uncovered the different underlying mechanisms of PD but the treatment of PD is still unknown due to the unavailability of effective therapies and preventive medicines in the current scenario. The pathophysiology and genetics of PD have been strongly associated with mitochondria in disease etiology. Several studies have investigated a complex molecular mechanism governing the identification and clearance of dysfunctional mitochondria from the cell, a mitochondrial quality control mechanism called mitophagy. Reduced mitophagy and mitochondrial impairment are found in both sporadic and familial PD. Pharmacologically modulating mitophagy and accelerating the removal of defective mitochondria are of common interest in developing a therapy for PD. However, despite the extensive understanding of the mitochondrial quality control pathway and its underlying mechanism, the therapeutic potential of targeting mitophagy modulation and its role in PD remains to be explored. Thus, targeting mitophagy using chemical agents and naturally occurring phytochemicals could be an emerging therapeutic strategy in PD prevention and treatment. We discuss the current research on understanding the role of mitophagy modulators in PD using Drosophila melanogaster as a model. We further explore the contribution of Drosophila in the pathophysiology of PD, and discuss comprehensive genetic analysis in flies and pharmacological drug screening to develop potential therapeutic molecules for PD.

Hyperthermia and doxorubicin release by Fol-LSMO nanoparticles induce apoptosis and autophagy in breast cancer cells.

Background: Studies on the anticancer effects of lanthanum strontium manganese oxide (LSMO) nanoparticles (NPs)-mediated hyperthermia at cellular and molecular levels are scarce. Materials & methods: LSMO NPs conjugated with folic acid (Fol-LSMO NPs) were synthesized, followed by doxorubicin-loading (DoxFol-LSMO NPs), and their effects on breast cancer cells were investigated. Results: Hyperthermia (45°C) and combination treatments exhibited the highest (∼95%) anticancer activity with increased oxidative stress. The involvement of intrinsic mitochondria-mediated apoptotic pathway and induction of autophagy was noted. Cellular and molecular evidence confirmed the crosstalk between apoptosis and autophagy, involving Beclin1, Bcl2 and Caspase-3 genes with free reactive oxygen species presence. Conclusion: The study confirmed hyperthermia and doxorubicin release by Fol-LSMO NPs induces apoptosis and autophagy in breast cancer cells.

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. Here we describe the generation of three transgenic lines mcherry-Atg8a, GFP-Ref(2)P and mito-roGFP2-Orp1 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.

Mitochondrial Redox Sensor for Drosophila Female Germline Stem Cells.

Mitochondrial reactive oxygen species (mROS), a major source of ROS within cells, functions as an important signaling molecule and has the ability to damage cellular macromolecules including DNA and proteins. Monitoring mROS levels is therefore essential to understand cell-cell communication and programmed cell death in all types of cell including stem cells. Here, we describe generation and characterization of a redox sensor for mROS that is specifically expressed in the germline stem cells (GSCs) in Drosophila. This redox sensor can be used to monitor the production of mROS and mitophagy in the GSCs during oogenesis.

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. Here, we describe 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.

Identification and characterization of the autophagy-related genes Atg12 and Atg5 in hydra.

Autophagy is an evolutionarily conserved process in eukaryotic cells that is involved in the degradation of cytoplasmic contents including organelles via the lysosome. Hydra is an early metazoan which exhibits simple tissue grade organization, a primitive nervous system, and is one of the classical non-bilaterian models extensively used in evo-devo research. Here, we describe the characterization of two core autophagy genes, Atg12 and Atg5, from hydra. In silico analyses including sequence similarity, domain analysis, and phylogenetic analysis demonstrate the conservation of these genes across eukaryotes. The predicted 3D structure of hydra Atg12 showed very little variance when compared to human Atg12 and yeast Atg12, whereas the hydra Atg5 predicted 3D structure was found to be variable, when compared with its human and yeast homologs. Strikingly, whole mount in situ hybridization showed high expression of Atg12 transcripts specifically in nematoblasts, whereas Atg5 transcripts were found to be expressed strongly in budding region and growing buds. This study may provide a framework to understand the evolution of autophagy networks in higher eukaryotes.

Fluorescent zinc(ii) complexes for gene delivery and simultaneous monitoring of protein expression.

Two new zinc(ii) complexes, [Zn(l-His)(NIP)]+(1) and [Zn(acac)2(NIP)](2) (where NIP is 2-(naphthalen-1-yl)-1H-imidazo[4,5-f][1,10]phenanthroline, acac = acetyl acetone), have been synthesized and characterized by elemental analysis, UV-vis, fluorescence, IR, 1H NMR and electron spray ionization mass spectroscopies. Gel retardation assay, atomic force microscopy and dynamic light scattering studies show that 1 and 2 can induce the condensation of circular plasmid pBR322 DNA into nanometer size particles under ambient conditions. Treatment of 2 with 5 mM EDTA restored 30% of the supercoiled form of DNA, revealing partial reversibility of DNA condensation. The in vitro transfection experiment demonstrates that the complexes can be used to deliver pCMV-tdTomato-N1 plasmid which expresses red fluorescent protein. The confocal studies show that the fluorescent nature of complexes is advantageous for visualizing the intracellular delivery of metal complexes as well as transfection efficiency using two distinct emission windows.

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. Here, we describe 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. Our data reveal that distinct autophagy programs are used by different cells within an animal, and disclose an unappreciated role for ubiquitin activation in autophagy.

Atg6 is required for multiple vesicle trafficking pathways and hematopoiesis in Drosophila.

Atg6 (beclin 1 in mammals) is a core component of the Vps34 complex that is required for autophagy. Beclin 1 (Becn1) functions as a tumor suppressor, and Becn1(+/-) tumors in mice possess elevated cell stress and p62 levels, altered NF-κB signaling and genome instability. The tumor suppressor function of Becn1 has been attributed to its role in autophagy, and the potential functions of Atg6/Becn1 in other vesicle trafficking pathways for tumor development have not been considered. Here, we generate Atg6 mutant Drosophila and demonstrate that Atg6 is essential for autophagy, endocytosis and protein secretion. By contrast, the core autophagy gene Atg1 is required for autophagy and protein secretion, but it is not required for endocytosis. Unlike null mutants of other core autophagy genes, all Atg6 mutant animals possess blood cell masses. Atg6 mutants have enlarged lymph glands (the hematopoietic organ in Drosophila), possess elevated blood cell numbers, and the formation of melanotic blood cell masses in these mutants is not suppressed by mutations in either p62 or NFκB genes. Thus, like mammals, altered Atg6 function in flies causes hematopoietic abnormalities and lethality, and our data indicate that this is due to defects in multiple membrane trafficking processes.

Regulation and function of autophagy during cell survival and cell death.

Autophagy is an important catabolic process that delivers cytoplasmic material to the lysosome for degradation. Autophagy promotes cell survival by elimination of damaged organelles and proteins aggregates, as well as by facilitating bioenergetic homeostasis. Although autophagy has been considered a cell survival mechanism, recent studies have shown that autophagy can promote cell death. The core mechanisms that control autophagy are conserved between yeast and humans, but animals also possess genes that regulate autophagy that are not present in yeast. These regulatory differences may be explained by the need to control autophagy in a cell context-specific manner in multicellular animals, such as during cell survival and cell death. Autophagy was thought to be a bulk cytoplasmic degradation mechanism, but recent studies have shown that specific cargo is recruited for degradation. This suggests the possibility that either cell survival or death may be regulated by selective autophagic clearance of cytoplasmic material. Here we summarize the mechanisms that regulate autophagy and how they may contribute to cell survival and death.

Autophagy as a trigger for cell death: autophagic degradation of inhibitor of apoptosis dBruce controls DNA fragmentation during late oogenesis in Drosophila.

Autophagy has been reported to contribute to cell death, but the underlying mechanisms remain largely unknown and controversial. We have: been studying oogenesis in Drosophila melanogaster as a model system to understand the interplay between autophagy and cell death. Using a novel autophagy reporter we found that autophagy occurs during developmental cell death of nurse cells in late oogenesis. Genetic inhibition: of autophagy-related genes atg1, atg13 and vps34 results in late-stage egg chambers containing persisting nurse cell nuclei without fragmented DNA and attenuation of caspase-3 cleavage. We found that Drosophila inhibitor of apoptosis dBruce is degraded by autophagy and this degradation promotes DNA fragmentation and subsequent nurse cell death. These studies demonstrate that autophagic degradation of an inhibitor: of apoptosis is a novel mechanism of triggering cell death.

Autophagic degradation of dBruce controls DNA fragmentation in nurse cells during late Drosophila melanogaster oogenesis.

Autophagy is an evolutionarily conserved pathway responsible for degradation of cytoplasmic material via the lysosome. Although autophagy has been reported to contribute to cell death, the underlying mechanisms remain largely unknown. In this study, we show that autophagy controls DNA fragmentation during late oogenesis in Drosophila melanogaster. Inhibition of autophagy by genetically removing the function of the autophagy genes atg1, atg13, and vps34 resulted in late stage egg chambers that contained persisting nurse cell nuclei without fragmented DNA and attenuation of caspase-3 cleavage. The Drosophila inhibitor of apoptosis (IAP) dBruce was found to colocalize with the autophagic marker GFP-Atg8a and accumulated in autophagy mutants. Nurse cells lacking Atg1 or Vps34 in addition to dBruce contained persisting nurse cell nuclei with fragmented DNA. This indicates that autophagic degradation of dBruce controls DNA fragmentation in nurse cells. Our results reveal autophagic degradation of an IAP as a novel mechanism of triggering cell death and thereby provide a mechanistic link between autophagy and cell death.

The role of Dpp and its inhibitors during eggshell patterning in Drosophila.

The Drosophila eggshell is patterned by the combined action of the epidermal growth factor [EGF; Gurken (Grk)] and transforming growth factor beta [TGF-beta; Decapentaplegic (Dpp)] signaling cascades. Although Grk signaling alone can induce asymmetric gene expression within the follicular epithelium, here we show that the ability of Grk to induce dorsoventral polarity within the eggshell strictly depends on Dpp. Dpp, however, specifies at least one anterior region of the eggshell in the absence of Grk. Dpp forms an anteriorposterior morphogen gradient within the follicular epithelium and synergizes with the dorsoventral gradient of Grk signaling. High levels of Grk and Dpp signaling induce the operculum, whereas lower levels of both pathways induce the dorsal appendages. We provide evidence that the crosstalk between both pathways occurs at least at two levels. First, Dpp appears to directly enhance the levels of EGF pathway activity within the follicular epithelium. Second, Dpp and EGF signaling collaborate in controlling the expression of Dpp inhibitors. One of these inhibitors is Drosophila sno (dSno), a homolog of the Ski/Sno family of vertebrate proto-oncogenes, which synergizes with daughters against dpp and brinker to set the posterior and lateral limits of the region, giving rise to dorsal follicle cells.

Molecular microbial diversity of a soil sample and detection of ammonia oxidizers from Cape Evans, Mcmurdo Dry Valley, Antarctica.

The aim of our study was to estimate the uncultured eubacterial diversity of a soil sample collected below a dead seal, Cape Evans, McMurdo, Antarctica by an SSU rDNA gene library approach. Our study by sequencing of clones from SSU rDNA gene library approach revealed high diversity in the soil sample from Antarctica. More than 50% of clones showed homology to Cytophaga-Flavobacterium-Bacteroides group; sequences also belonged to alpha, beta, gamma proteobacteria, Thermus-Deinococcus and high GC gram-positive group; Phylogenetic analysis of the SSU rDNA clones showed the presence of species belonging to Cytophaga spp., Vitellibacter vladivostokensis, Aequorivita lipolytica, Aequorivita crocea, Flavobacterium spp., Flexibacter sp., Subsaxibacter broadyi, Bacteroidetes, Roseobacter sp., Sphingomonas baekryungensis, Nitrosospira sp., Nitrosomonas cryotolerans, Psychrobacter spp., Chromohalobacter sp., Psychrobacter okhotskensis, Psychrobacter fozii, Psychrobacter urativorans, Rubrobacter radiotolerans, Marinobacter sp., Rubrobacteridae, Desulfotomaculum aeronauticum and Deinococcus sp. The presence of ammonia oxidizing bacteria in Antarctica soil was confirmed by the presence of the amoA gene. Phylogenetic analysis revealed grouping of clones with their respective groups.