Autophagy functions in programmed cell death

Autophagic cell death is a prominent morphological form of cell death that occurs in diverse animals. Autophagosomes are abundant during autophagic cell death, yet the functional role of autophagy in cell death has been enigmatic. We find that autophagy and the Atg genes are required for autophagic cell death of Drosophila salivary glands. Although caspases are present in dying salivary glands, autophagy is required for complete cell degradation. Further, induction of high levels of autophagy results in caspase-independent autophagic cell death. Our results provide the first in vivo evidence that autophagy and the Atg genes are required for autophagic cell death and confirm that autophagic cell death is a physiological death program that occurs during development.     

Autophagy in the control of programmed cell death

Programmed cell death (PCD) is essential for plant development and immunity. Localized PCD is associated with the hypersensitive response (HR), which is a constituent of a successful plant innate immune response. Plants have developed mechanisms to meticulously prevent HR-PCD lesions from spreading. Our understanding of these mechanisms is still in its incipient stages. A recent study demonstrated that autophagy, a universally conserved process of macromolecule turnover, plays a pivotal role in controlling HR-PCD. The molecular identity of the mediators between the PCD and HR pathways is still obscure, but recent work has begun to shed light on the relationship between HR-PCD and autophagy and to suggest possible mechanisms for the regulation of these pathways.     

Autophagy activity contributes to programmed cell death in Caenorhabditis elegans

The physiological relationship between autophagy and programmed cell death during C. elegans development is poorly understood. In C. elegans, 131 somatic cells and a large number of germline cells undergo programmed cell death. Autophagy genes function in the removal of somatic cell corpses during embryogenesis. Here we demonstrated that autophagy activity participates in germ-cell death induced by genotoxic stress. Upon γ ray treatment, fewer germline cells execute the death program in autophagy mutants. Autophagy also contributes to physiological germ-cell death and post-embryonic cell death in ventral cord neurons when ced-3 caspase activity is partially compromised. Our study reveals that autophagy activity contributes to programmed cell death during C. elegans development.     

Signaling unmasked: Autophagy and catalase promote programmed cell death

Autophagy contributes to the removal of harmful cellular refuse, whereas catalase plays an important protective role by detoxifying reactive oxygen species. We recently found that autophagy and catalase are also required for promoting programmed cell death induced during plant immune responses. Here we discuss the difficulties in identifying cell death effectors, which are also required to maintain cellular homeostasis, and how their prodeath roles were unmasked using an unbiased forward genetics approach.     

Autophagy regulates programmed cell death during the plant innate immune response

The plant innate immune response includes the hypersensitive response (HR), a form of programmed cell death (PCD). PCD must be restricted to infection sites to prevent the HR from playing a pathologic rather than protective role. Here we show that plant BECLIN 1, an ortholog of the yeast and mammalian autophagy gene ATG6/VPS30/beclin 1, functions to restrict HR PCD to infection sites. Initiation of HR PCD is normal in BECLIN 1-deficient plants, but remarkably, healthy uninfected tissue adjacent to HR lesions and leaves distal to the inoculated leaf undergo unrestricted PCD. In the HR PCD response, autophagy is induced in both pathogen-infected cells and distal uninfected cells; this is reduced in BECLIN 1-deficient plants. The restriction of HR PCD also requires orthologs of other autophagy-related genes including PI3K/VPS34, ATG3, and ATG7. Thus, the evolutionarily conserved autophagy pathway plays an essential role in plant innate immunity and negatively regulates PCD.     

Autophagy,programmed cell death and reactive oxygen species in sexual reproduction in plants

Autophagy is one of the major cellular processes of recycling of proteins, metabolites and intracellular organelles, and plays crucial roles in the regulation of innate immunity, stress responses and programmed cell death (PCD) in many eukaryotes. It is also essential in development and sexual reproduction in many animals. In plants, although autophagy-deficient mutants of Arabidopsis thaliana show phenotypes in abiotic and biotic stress responses, their life cycle seems normal and thus little had been known until recently about the roles of autophagy in development and reproduction. Rice mutants defective in autophagy show sporophytic male sterility and immature pollens, indicating crucial roles of autophagy during pollen maturation. Enzymatic production of reactive oxygen species (ROS) by respiratory burst oxidase homologues (Rbohs) play multiple roles in regulating anther development, pollen tube elongation and fertilization. Significance of autophagy and ROS in the regulation of PCD of transient cells during plant sexual reproduction is discussed in comparison with animals.     

Autophagy promotes programmed cell death and corpse clearance in specific cell types of the Arabidopsis root cap

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Erythrocyte programmed cell death

Eryptosis, the suicidal death of erythrocytes, is characterised by cell shrinkage, membrane blebbing and cell membrane phospholipid scrambling with phosphatidylserine exposure at the cell surface. Phosphatidylserine-exposing erythrocytes are recognised by macrophages, which engulf and degrade the affected cells. Reported triggers of eryptosis include osmotic shock, oxidative stress, energy depletion, ceramide, prostaglandin E(2), platelet activating factor, hemolysin, listeriolysin, paclitaxel, chlorpromazine, cyclosporine, methylglyoxal, amyloid peptides, anandamide, Bay-5884, curcumin, valinomycin, aluminium, mercury, lead and copper. Diseases associated with accelerated eryptosis include sepsis, malaria, sickle-cell anemia, beta-thalassemia, glucose-6-phosphate dehydrogenase (G6PD)-deficiency, phosphate depletion, iron deficiency, hemolytic uremic syndrome and Wilsons disease. Eryptosis may be inhibited by erythropoietin, adenosine, catecholamines, nitric oxide (NO) and activation of G-kinase. Most triggers of eryptosis except oxidative stress are effective without activation of caspases. Their signalling involves formation of prostaglandin E(2) with subsequent activation of cation channels and Ca2+ entry and/or release of platelet activating factor (PAF) with subsequent activation of sphingomyelinase and formation of ceramide. Ca2+ and ceramide stimulate scrambling of the cell membrane. Ca2+ further activates Ca2+-sensitive K+ channels leading to cellular KCl loss and cell shrinkage and stimulates the protease calpain resulting in degradation of the cytoskeleton. Eryptosis allows defective erythrocytes to escape hemolysis. On the other hand, excessive eryptosis favours the development of anemia. Thus, a delicate balance between proeryptotic and antieryptotic mechanisms is required to maintain an adequate number of circulating erythrocytes and yet avoid noneryptotic death of injured erythrocytes.     

Autophagy and cell death

Autophagy exerts dual functions in cancer, acting as both a tumor suppressor, for example, by preventing the accumulation of damaged proteins and organelles, and as a tumor promoter that supports tumor growth. Many anticancer therapies engage autophagy as part of a cellular response. However, the question of whether or not autophagic activity in cells undergoing cell death is the cause of death or whether it is actually an attempt to support survival in response to cellular stress conditions has been discussed with great controversy.     

Microgravity-induced programmed cell death in astrocytes

Cultured astrocytes were submitted to simulated microgravity using a Fokker clinostat under continuous rotation (60 rpm) for 15', 30', 1h, 20h and 32h. Samples processing included (i) nuclear stainings using Propidium Iodide and 4,6-diamidino-2-phenilindole, dihydro chloride, (ii) immunohistochemical identification of Caspase-7, (iii) identification of DNA fragmentation using the terminal dUTP nick end labelling and (iv) Scanning Electron Microscope analysis. After 30' at simulated microgravity the glial cells showed morphological evidence of apoptosis: cell shrinkage, chromatin condensation, nuclear blebs and fragmentation. The enzyme caspase-7 was present and DNA fragmentation was evident. After 32h the density of the cell population was much lower than that observed in controls.     

Developmental programmed cell death in plants

Mechanisms of plant developmental programmed cell death (PCD) have been intensively studied in recent years. Most plant developmental PCD is triggered by plant hormones, and the 'death signal' may be transduced by hormonal signaling pathways. Although there are some fundamental differences in the regulation of developmental PCD in various eukaryotes of different kingdoms, hormonal control and death signal transduction via pleiotropic signaling pathways constitute a common framework. However, plants possess a unique process of PCD execution that depends on vacuolar lytic function. Comparisons of the developmental PCD mechanisms of plants and other organisms are providing important insights into the detailed characteristics of developmental PCD in plants.     

Developmentally programmed cell death in Drosophila

During the development of metazoans, programmed cell death (PCD) is essential for tissue patterning, removal of unwanted cells and maintaining homeostasis. In the past 20 years Drosophila melanogaster has been one of the systems of choice for studies involving developmental cell death, providing an ideal genetically tractable model of intermediary complexity between Caenorhabditis elegans and mammals. The lessons learned from studies using Drosophila indicate both the conserved nature of the many cell death pathways as well as novel and unexpected mechanisms. In this article we review the understanding of PCD during Drosophila development, highlighting the key mechanisms that are evolutionarily conserved as well as apparently unusual pathways, which indicate divergence, but provide evidence of complexity acquired during organismic evolution. This article is part of a Special Section entitled: Cell Death Pathways. Guest Editors: Frank Madeo and Slaven Stekovic.     

Autophagy in cell survival and death

Macroautophagy hereafter referred to as autophagy is a major lysosomal catabolic pathway for macromolecules and organelles conserved in eukaryotic cells. The discovery of the molecular basis of autophagy has uncovered its importance during development, life extension and in pathologies such as cancer, certain forms of myopathies and neurodegenerative diseases. Autophagy is a cell survival mechanism during starvation that is controlled by amino acids. Starvation-induced autophagy is an anti-apoptotic mechanism. However autophagy is also an alternative to apoptosis through autophagic cell death. In many situations apoptosis and autophagy can both contribute to cell dismantlement.     

Caspase function in programmed cell death

The first proapoptotic caspase, CED-3, was cloned from Caenorhabditis elegans in 1993 and shown to be essential for the developmental death of all somatic cells. Following the discovery of CED-3, caspases have been cloned from several vertebrate and invertebrate species. As reviewed in other articles in this issue of Cell Death and Differentiation, many caspases function in nonapoptotic pathways. However, as is clear from the worm studies, the evolutionarily conserved role of caspases is to execute programmed cell death. In this article, I will specifically focus on caspases that function primarily in cell death execution. In particular, the physiological function of caspases in apoptosis is discussed using examples from the worm, fly and mammals.     

Chitosan-induced programmed cell death in plants

Chitosan, CN⁻, or H₂O₂ caused the death of epidermal cells (EC) in the epidermis of pea leaves that was detected by monitoring the destruction of cell nuclei; chitosan induced chromatin condensation and marginalization followed by the destruction of EC nuclei and subsequent internucleosomal DNA fragmentation. Chitosan did not affect stoma guard cells (GC). Anaerobic conditions prevented the chitosan-induced destruction of EC nuclei. The antioxidants nitroblue tetrazolium or mannitol suppressed the effects of chitosan, H₂O₂, or chitosan + H₂O₂ on EC. H₂O₂ formation in EC and GC mitochondria that was determined from 2′,7′-dichlorofluorescein fluorescence was inhibited by CN⁻ and the protonophoric uncoupler carbonyl cyanide m-chlorophenylhydrazone but was stimulated by these agents in GC chloroplasts. The alternative oxidase inhibitors propyl gallate and salicylhydroxamate prevented chitosan- but not CN⁻-induced destruction of EC nuclei; the plasma membrane NADPH oxidase inhibitors diphenylene iodonium and quinacrine abolished chitosan- but not CN⁻-induced destruction of EC nuclei. The mitochondrial protein synthesis inhibitor lincomycin removed the destructive effect of chitosan or H₂O₂ on EC nuclei. The effect of cycloheximide, an inhibitor of protein synthesis in the cytoplasm, was insignificant; however, it was enhanced if cycloheximide was added in combination with lincomycin. The autophagy inhibitor 3-methyladenine removed the chitosan effect but exerted no influence on the effect of H₂O₂ as an inducer of EC death. The internucleosome DNA fragmentation in conjunction with the data on the 3-methyladenine effect provides evidence that chitosan induces programmed cell death that follows a combined scenario including apoptosis and autophagy. Based on the results of an inhibitor assay, chitosan-induced EC death involves reactive oxygen species generated by the NADPH oxidase of the plasma membrane.     

Apoptotic-like programmed cell death in plants

Programmed cell death (PCD) is now accepted as a fundamental cellular process in plants. It is involved in defence, development and response to stress, and our understanding of these processes would be greatly improved through a greater knowledge of the regulation of plant PCD. However, there may be several types of PCD that operate in plants, and PCD research findings can be confusing if they are not assigned to a specific type of PCD. The various cell-death mechanisms need therefore to be carefully described and defined. This review describes one of these plant cell death processes, namely the apoptotic-like PCD (AL-PCD). We begin by examining the hallmark 'apoptotic-like' features (protoplast condensation, DNA degradation) of the cell's destruction that are characteristic of AL-PCD, and include examples of AL-PCD during the plant life cycle. The review explores the possible cellular 'executioners' (caspase-like molecules; mitochondria; de novo protein synthesis) that are responsible for the hallmark features of the cellular destruction. Finally, senescence is used as a case study to show that a rigorous definition of cell-death processes in plant cells can help to resolve arguments that occur in the scientific literature regarding the timing and control of plant cell death.