Autophagic Death Research Articles

Steroid Regulation of Midgut Cell Death during Drosophila Development

Steroid hormones trigger dynamic tissue changes during animal development by activating cell proliferation, cell differentiation, and cell death. Here we characterize steroid regulation of changes in midgut structure during the onset of Drosophila metamorphosis. Following an increase in the steroid 20-hydroxyecdysone (ecdysone) at the end of larval development, future adult midgut epithelium is formed, and the larval midgut is rapidly destroyed. Mutations in the steroid-regulated genes BR-C and E93 differentially impact larval midgut cell death but do not affect the formation of adult midgut epithelia. In contrast, mutations in the ecdysone-regulated E74A and E74B genes do not appear to perturb midgut development during metamorphosis. Larval midgut cells possess vacuoles that contain cellular organelles, indicating that these cells die by autophagy. While mutations in the BR-C, E74, and E93 genes do not impact DNA degradation during this cell death, mutations in BR-C inhibit destruction of larval midgut structures, including the proventriculus and gastric caeca, and E93 mutants exhibit decreased formation of autophagic vacuoles. Dying midguts express the rpr, hid, ark, dronc, and crq cell death genes, suggesting that the core cell death machinery is involved in larval midgut cell death. The transcription of rpr, hid, and crq are altered in BR-C mutants, and E93 mutants possess altered transcription of the caspase dronc, providing a mechanism for the disruption of midgut cell death in these mutant animals. These studies indicate that ecdysone triggers a two-step hierarchy composed of steroid-induced regulatory genes and apoptosis genes that, in turn, regulate the autophagic death of midgut cells during development.

Autophagy and Receptor-Induced Neurodegeneration

Much attention has focused on the role of apoptosis in neurodegenerative diseases. Yue et al. present evidence that an alternative form of death--characterized by excessive autophagy--may also be a critical factor. In autophagic death, the cytoplasmic matrix and organelles are engulfed in autophagosomes and autophagic vacuoles and degraded in lysosomes. Mice that have a mutation in the glutamate receptor subunit GluRδ2 have constitutively active cation-permeable glutamate receptor channels and are called lurcher mice--so-named for their ataxic behavior, which results from degeneration of the cerebellar cortex. Yue et al. used a yeast two-hybrid screen to find proteins that interacted with the cytoplasmic tail of the GluRδ2 subunit and identified a protein called nPIST. The nPIST protein has properties that suggest it functions as an adaptor, possibly mediating protein-protein interactions at the receptor. A second two-hybrid screen, this time with coiled-coil domains of PIST, identified Beclin1, a protein previously implicated in control of membrane dynamics during autophagy. When transfected into cultured 293 cells, nPIST that lacked its PDZ domain (a likely site for interaction with other proteins) synergized with Beclin 1 to cause autophagy. Autophagy was also detected in dying Purkinje cells in the cerebellum of lurcher mice. The results suggest that inappropriate activation of glutamate receptors may lead to autophagic neurodegeneration through signals mediated by proteins clustered at the receptor, including nPIST and Beclin 1. Intriguingly, Beclin1 has also been shown to associate with members of the Bcl2 family of proteins, which regulate apoptosis. The authors note that inclusion of Bcl2 family members in a complex with the GluRδ2 subunit could provide coordinated regulation of both autophagy and apoptosis in the compromised Purkinje cells of lurcher mice. Z. Yue, A. Horton, M. Bravin, P. L. DeJager, F. Selimi, N. Heintz, A novel protein complex linking the δ2 glutamate receptor and autophagy: Implications for neurodegeneration in lurcher mice, Neuron 35 , 921-933 (2002). [Online Journal]

A Novel Protein Complex Linking the δ2 Glutamate Receptor and Autophagy: Implications for Neurodegeneration in Lurcher Mice

Autophagy is a pathway for bulk degradation of subcellular constituents that is hyperactivated in many neurodegenerative conditions. It has been considered a second form of programmed cell death. Death of cerebellar Purkinje cells in lurcher animals is due to a mutation in GluRδ2 that results in its constitutive activation. Here we have identified protein interactions between GluRδ2, a novel isoform of a PDZ domain-containing protein (nPIST) that binds to this receptor, and Beclin1. nPIST and Beclin1 can synergize to induce autophagy. GluRδ2 Lc, but not GluRδ2 wt, can also induce autophagy. Furthermore, dying lurcher Purkinje cells contain morphological hallmarks of autophagic death in vivo. These results provide strong evidence that a direct link exists between GluRδ2 Lc receptor and stimulation of the autophagic pathway in dying lurcher Purkinje cells.

An ARL1 mutation affected autophagic cell death in yeast, causing a defect in central vacuole formation.

When the cdc28 strain of Saccharomyces cerevisiae is incubated at restrictive temperatures, the yeasts digest themselves in 7 days by activating autophagic machinery. In parallel, the cell-proliferative activity decreases progressively after about 48 h. We have previously referred to this phenomenon as autophagic death. In the present study, we isolated and characterized a recessive mutant strain, dlp2, which delays the progression toward autophagic death. The cdc28 dlp2 cells contain many small vesicles instead of the large central vacuoles that are usually found in parental cdc28 cells. We showed that the dlp2 phenotype results from the presence of a single mutation in the gene ARL1 (ADP-ribosylation factor-like protein 1). Morphological and biochemical analyses of cdc28 dlp2 suggested that a defect in central vacuole formation is caused by aberrant membrane trafficking, although the protein-sorting to vacuoles is not affected. After a shift to a restrictive temperature, the components of the cytoplasm and nucleus of cdc28 dlp2 were condensed, with an accompanying formation of vesicles in the periphery (epiplasm) of the cells rather than an activation of the autophagic machinery. Introducing this ARL1 mutation into the normal ARL1 locus of the wild-type W303 strain again inhibited the progression of apoptotic cell death due to a defect in vacuole formation, which in this case was induced by the proapoptotic protein Bax. Thus, the ARL1 gene plays an important role in the formation of central vacuoles and in the progression of programmed cell death induced by cell-cycle arrest or Bax. These results suggested the presence of a programmed-cell death machinery in yeast that is similar to that related to the Type II cell death of mammalian cells characterized by autophagocytosis.

Mammary Gland Cell Death Also Involves Lysosomal Autophagy

The mammary gland undergoes apoptosis when estrogen ablation occurs, either naturally or enforced. The gland is known to execute the apoptotic process post weaning. Although the involuting mammary gland displays the characteristic biochemical features of apoptosis, including DNA fragmentation, chromatin condensation, and the formation of apoptotic bodies, it also shows evidence of an autophagic death. In this report, apoptosis of the gland was induced by removing the pups from their nursing mothers. In particular, we show that lysosomes increased in size and number, and moved from basal to apical regions in dying rat mammary gland cells. Lysosomal enzyme activities were significantly greater in degenerating mammary gland (day 4 post weaning) epithelial cells when compared with day 0 gland cells. Moreover, these hydrolases were responsible for degrading cytosolic and nuclear components, and thus the whole cell. Taken together, our results demonstrate that the mammary gland dies by lysosomal autophagy in addition to apoptosis during post-lactational involution. Our studies indicate that the lysosomal compartment may serve as an important target organelle for the creation of specific, effective, and novel therapies for breast cancer.

Steroid-triggered death by autophagy.

Programmed cell death is a critical part of normal development, removing obsolete tissues or cells and sculpting body parts to assume their appropriate form and function. Most programmed cell death occurs by apoptosis of individual cells or autophagy of groups of cells. Although these pathways have distinct morphological characteristics, they also have a number of features in common, suggesting some overlap in their regulation. A recent paper by Lee and Baehrecke provides further support for this proposal.(1) These authors present, for the first time, a genetic analysis of autophagy, using the steroid-triggered metamorphosis of Drosophila as a model system. They demonstrate a remarkable degree of overlap between the control of apoptosis and autophagy as well as a key role for the steroid-inducible gene E93 in directing the autophagic death response. This paper also shows that E93 can direct cell death independently from the known death-inducer genes, defining a novel death pathway in Drosophila.

Meeting report: mechanisms of cell death 2000.

“Truth does not become more true by virtue of the fact that the entire world agrees with it, nor less so even if the whole world disagrees with it” (Maimonides). This quotation best expresses the theme of the third biennial meeting of the International Cell Death Society held on May 6, 2000 in Escorial, Spain. The meeting entitled “Mechanisms of Cell Death,” organized by Zahra Zakeri, Richard A. Lockshin, and Carlos Martinez-A. It filled to capacity with over 300 participants. The speakers included both junior (55%) and senior (45%) scientists with 42% women. The broad perspective covered six sections: Development and Aging; Extracellular Signaling; Cell Death in Disease; Cellular Signaling; Cell Survival; and Proteolysis, ending with a workshop on phagocytosis. In this meeting many of the speakers either challenged ideas that recently dominated the field, or elaborated and nuanced ideas that previously were considered to be straightforward. For instance, initiation of cell death remains as complex a question as ever. Numerous promoter sites for the Drosophila gene reaper indicate very complex regulation (Abrams). In AIDS, there is a complex balance between host-driven pro-apoptotic and virus-driven antiapoptotic forces (Cossarizza) and a dysregulation of cytokines (Gougeon). The role of mitochondria in apoptosis was addressed at several levels. Kroemer demonstrated that Bcl2 could inhibit a liposome-embedded Permeability Transition Pore Complex. Martinou considered that Bax can release cytochrome c but also can act by a secondary mechanism. It was reported that at the cellular level Bcl2 and bax differed in their capacities to localize to different compartments, indicating that their behavior and hence functions in intact cells may differ from strictly biochemical measurements in vitro (Borner). Physical, autophagic, loss of mitochondria was argued by Tolkovsky as a death commitment point. Depolarization of only a subset of mitochondria could initiate the apoptotic program. (D’Herde). An intriguing change of view was reflected in the mode of activation and function of the caspases, and the relationship of caspase-dependent to caspase-independent death. Several researchers suggested functions other than apoptosis for caspases. Hardwick found that proteolysis converted anti-apoptotic proteins into pro-apoptotic proteins, thus guaranteeing the launching of apoptosis. Roy contended that the dormancy of caspases is controlled by a DDD (Asp-Asp-Asp) “safety catch” sequence in caspases. Caspase 8 was shown to correlate inversely with tumor radiosensitivity (Zhivotovsky). FLIP inhibits casp8 (Thome) and HSP72 blocks casp3 activation (Anderson). Jaatella reported the intriguing finding that depletion of HSP70 (by antisense mRNA) activated a tumor-specific death program that was independent of caspases and bypassed bcl-2. Evidence was presented for the function of caspases in roles other than in cell death. Apaf1 and caspase 9 are important in tumor development (Soengas). Woo, looking at casp3 knockouts, demonstrated a role for casp3 in proliferation, perhaps by downregulating p21. Silke, using a novel screen in yeast, found that XIAPs inhibit UV-induced apoptosis, a surprising finding because they were supposed to act at a far downstream point. Other proteases such as matrix metaloproteinases and stromelysin were posited to be an extremely important part of autophagic death in tadpole metamorphosis (Shi; also noted by Linden) and granzyme B can produce reactive oxygen substances and ψm without the participation of caspases (Greenberg). More evidence for the involvement of other proteases was presented by Torriglia, who showed that, in lens, cleavage of leukocyte elastase inhibitor (LEI) achieved a double purpose: it relieved inhibition of a serine protease; and it turned LEI into L-DNase II, which is involved in degradation of the DNA. Roberg found that, in oxidative stress-induced apoptosis, lysosomal destabilization preceded mitochondrial changes, and pepstatin, an inhibitor of cathepsin D, blocked progress of apoptosis. Bursch, arguing that autophagic death differed from chemical apoptosis, emphasized that autophagic death requires ATP, is specific for elimination of different types of organelles, and requires extensive involvement of the cytoskeleton. The meeting was one of the first to emphasize phagocytosis, both in the primary meeting and in the following

The DLP1 mutant of the yeast Saccharomyces cerevisiae with an increased copy number of the 2μ plasmid shows a shortened lifespan

We isolated and characterized a recessive mutant, named dlp1, which shows the Dlp phenotype (delayed loss of proliferation activity) during the autophagic death of cdc28. The dlp1 mutant was found to consist of two subtypes of cells based on colony morphology. One subtype with the Dlp phenotype, named dlp1-l, became large, red, and nibbled during the incubation, suggesting that the cells on the surface of the colonies were dying. The other without the Dlp phenotype, named dlp1-s, retained small, white colonies even after a prolonged incubation and was found to be a petite mutant. The change from dlp1-l to dlp1-s (petite) occurred much more frequently (about 15%) than that from the wild-type to petite mutant (less than 1%). The lifespan of both subtypes of cells was severely shortened. The copy number of the endogenous 2μ plasmid of dlp1-l was 68-fold that of the original cdc28, and decreased by half after the conversion to dlp1-s (petite). A 4.0-kbp fragment of the 2μ plasmid containing REP2 decreased the copy number of the endogenous 2μ plasmid to 8-fold that of the original cdc28 cells and partially rescued the shortened lifespan, in addition to resulting in the complete complementation of the Dlp and nibbled-colony phenotypes. These results suggest that DLP1 is a chromosomal gene that regulates the copy number of the 2μ plasmid, and that the shortening of the lifespan and other effects of the dlp1 mutation are likely caused by the increased copy number of the endogenous 2μ plasmid.

Transient c- fos expression accompanies naturally occurring cell death in the developing interhemispheric cortex of the rat

We have searched for the possible correlation of naturally occurring cell death with spontaneously enhanced c- fos expression in the developing cerebral cortex of normal Wistar albino rats. During the late prenatal and early postnatal period, cells with irregular contours and intracytoplasmic electron-dense granules (granule-containing cells) were apparent in the interhemispheric cortex, including the anterior cingulate and the retrosplenial cortices. These cells were loosely packed within the cortical layers derived from the cortical plate. Having excluded the possibility that these cells could be phagocytes by immunocytochemical experiments, we propose that they are cells in different phases of a process of autophagic degeneration and death. Images of extreme nuclear pyknosis were also apparent in identical locations. Cells showing immunoreactivity for c-Fos protein appeared in the same cortical areas. The immunoreactive cells were very abundant in the retrosplenial cortex, but were also present in the anterior cingulate cortex. These cells showed markedly irregular contours and large, densely immunoreactive intracytoplasmic inclusions; these images were similar to those of granule-containing cells revealed by conventional stains. The immunoreactivity for c-Fos protein was ephemeral, occurring exclusively during embryonic days 20 and 21, but granule-containing cells were observed for a longer period. The present results provide evidence, albeit indirect, that c- fos expression may occur in certain neural cells at the onset of a process of death by autophagia, and suggest a possible involvement of the proto-oncogene c- fos in certain forms of naturally occurring neuronal death.