Apoptosome impairment during development results in activation of an autophagy program in cerebral cortex

The deficiency of upstream regulators of the mitochondrial death pathway has been recently shown to trigger in vitro a cellular process of self-clearance with features of autophagy. We show here that, when Apaf1 (responsible for apoptosome formation) is downregulated in vivo in cortical precursors, cells express markers of neuronal differentiation, accumulate in ectopic cortical masses and show hallmarks of the beclin-1-dependent pathway of autophagy, probably activated by a depletion in growth factors in the cells' microenvironment. To visualize this process in a cell culture model system, we also used a neural precursor cell line to mimic growth factor starvation in the absence of the apoptosome and tracked autophagolysosome formation. Our findings demonstrate the existence of an interplay between the autophagy and apoptosis pathways in vivo in brain development, and possibly link the absence of apoptosis to the occurrence of pathological conditions associated with peculiar cellular morphotypes.     

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 can be a killer even in apoptosis-competent cells

Despite abundant evidence for autophagic cell death as a morphological type, the notion that autophagy can actually contribute mechanistically to the cell's death is controversial. In cells capable of apoptosis, autophagic cell death has been dismissed by some authors as a morphologically unusual form of apoptosis. But strong recent evidence for autophagy-mediated death of cells rendered incapable of apoptosis has been criticized on the grounds that this cell death is too artificial to be relevant to normal cells. We here argue from our own and other recent evidence that autophagy can mediate the death even of apoptosis-competent cells.     

Steroid regulation of autophagic programmed cell death during development

Apoptosis and autophagy are morphologically distinct forms of programmed cell death. While autophagy occurs during the development of diverse organisms and has been implicated in tumorigenesis, little is known about the molecular mechanisms that regulate this type of cell death. Here we show that steroid-activated programmed cell death of Drosophila salivary glands occurs by autophagy. Expression of p35 prevents DNA fragmentation and partially inhibits changes in the cytosol and plasma membranes of dying salivary glands, suggesting that caspases are involved in autophagy. The steroid-regulated BR-C, E74A and E93 genes are required for salivary gland cell death. BR-C and E74A mutant salivary glands exhibit vacuole and plasma membrane breakdown, but E93 mutant salivary glands fail to exhibit these changes, indicating that E93 regulates early autophagic events. Expression of E93 in embryos is sufficient to induce cell death with many characteristics of apoptosis, but requires the H99 genetic interval that contains the rpr, hid and grim proapoptotic genes to induce nuclear changes diagnostic of apoptosis. In contrast, E93 expression is sufficient to induce the removal of cells by phagocytes in the absence of the H99 genes. These studies indicate that apoptosis and autophagy utilize some common regulatory mechanisms.     

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.

Addendum to: Berry DL, Baehrecke EH. Growth arrest and autophagy are required for programmed salivary gland cell degradation in Drosophila. Cell 2007; 131:1137-48.     

Physiological and pathological roles of Apaf1 and the apoptosome

Different cellular pathways can lead to apoptosis. Apaf1 is the molecular core of the apoptosome, a multiproteic complex mediating the so-called mitochondrial pathway of cell death. The importance of this pathway during development has been clearly demonstrated by knocking out key genes. Also, the relevance of Apaf1 dosage during development has been recently underlined. Moreover, a growing body of evidences seems to point out a possible role of the mitochondria-dependent apoptosis in different pathologies. In particular, we discuss here some recent evidences regarding the putative role of the apoptosome in neurodegeneration and cancer.     

Real-time observation of autophagic programmed cell death of<Emphasis Type="Italic"> Drosophila</Emphasis> salivary glands in vitro

Autophagy, a form of programmed cell death (PCD) that is morphologically distinguished from apoptosis, is thought to be as prevalent as apoptosis, at least during development. In insect metamorphosis, the steroid hormone 20-hydroxyecdysone (ecdysone) activates autophagic PCD to eliminate larval structures that are no longer needed. However, in comparison with apoptosis, there are not many studies on the regulation mechanisms of autophagy. To provide a useful model for studying autophagic PCD, I established an in vitro culture system that enables real-time observation of the autophagic cell destruction of Drosophila salivary glands. The new system revealed that de novo gene expression was still required for the destruction of salivary glands dissected from phanerocephalic pupae. This indicates the usefulness of the system for exploring genes that participate in the last processes of autophagic PCD.Edited by N. Satoh     

Apaf1 and the apoptotic machinery

The molecular characterization of the Caenorhabditis elegans cell death genes has been crucial in revealing some of the biochemical mechanisms underlying apoptosis in all animals. Four C. elegans genes, egl-1, ced-9, ced-4 and ced-3 are required for all somatic programmed cell death to occur. This genetic network is highly conserved during evolution. The pro-death gene egl-1 and the anti-death gene ced-9 have structural and functional similarities to the vertebrate Bcl2 gene family. The killer gene ced-3 encodes a cystein-aspartate protease (caspase), which is the archetype of a family of conserved proteins known as effectors of apoptosis in mammals. Zou and collaborators1 reported the biochemical identification of an apoptotic protease activating factor (Apaf1), a human homolog of C. elegans CED-4, providing important clues to how CED-4 and its potential relatives could work. A number of proteins have been shown to interact with Apaf1 or to be determinant for its activity as an apoptotic adapter. The aim of this review is to provide an overview of the recent progress made in the field of developmental apoptosis by means of the murine Apaf1 targeted mutations. The central role of Apaf1 in the cell death machinery (apoptosome) and its involvement in different apoptotic pathways will also be discussed.     

Caspase-independent cell death and mitochondrial disruptions observed in the Apaf1-deficient cells

Apaf1 is a critical molecule in the mitochondria-dependent apoptotic pathway. Here we show that Apaf1-deficient embryonic fibroblasts died at a later phase of apoptotic induction, although these cells were resistant to various apoptotic stimulants at an early phase. Neither caspase 3 activation nor nuclear condensation was observed during this cell death of Apaf1-deficient cells. Electron microscopic examination revealed that death in response to apoptotic stimulation resembled necrosis in that nuclei were round and swollen with low electron density. Necrosis-like cell death was also observed in wild-type cells treated with z-VAD-fmk. Mitochondria were not only morphologically abnormal but functionally affected, since mitochondrial transmembrane potential (DeltaPsim) was lost even in cells with intact plasma membrane integrity. These mitochondrial alterations were also observed in the wild-type cells dying of apoptosis. Combined, these data suggest that cells without caspase activation, such as Apaf1-deficient cells or cells treated with caspase inhibitors, die of necrosis-like cell death with mitochondrial damage in response to "apoptotic stimulation."     

Autophagy Can Be a Killer Even in Apoptosis-Competent Cells

Despite abundant evidence for autophagic cell death as a morphological type, the notion that autophagy can actually contribute mechanistically to the cell's death is controversial. In cells capable of apoptosis, autophagic cell death has been dismissed by some authors as a morphologically unusual form of apoptosis. But strong recent evidence for autophagy-mediated death of cells rendered incapable of apoptosis has been criticized on the grounds that this cell death is too artificial to be relevant to normal cells. We here argue from our own and other recent evidence that autophagy can mediate the death even of apoptosis-competent cells.

Addendum to:

Role of Phosphoinositide 3-Kinase in the Autophagic Death of Serum-Deprived PC12 Cells.

A. Guillon-Munos, M.X.P. van Bemmelen and P.G.H. Clarke

Apoptosis 2005; 10:1031-41.     

Autophagy gene disruption reveals a non-vacuolar cell death pathway in Dictyostelium

Types of cell death include apoptosis, necrosis, and autophagic cell death. The latter can be defined as death of cells containing autophagosomes, autophagic bodies, and/or vacuoles. Are autophagy and vacuolization causes, consequences, or side effects in cell death with autophagy? Would control of autophagy suffice to control this type of cell death? We disrupted the atg1 autophagy gene in Dictyostelium discoideum, a genetically tractable model for developmental autophagic vacuolar cell death. The procedure that induced autophagy, vacuolization, and death in wild-type cells led in atg1 mutant cells to impaired autophagy and to no vacuolization, demonstrating that atg1 is required for vacuolization. Unexpectedly, however, cell death still took place, with a non-vacuolar and centrally condensed morphology. Thus, a cell death mechanism that does not require vacuolization can operate in this cell death model showing conspicuous vacuolization. The revelation of non-vacuolar cell death in this protist by autophagy gene disruption is reminiscent of caspase inhibition revealing necrotic cell death in animal cells. Thus, hidden alternative cell death pathways may be found across kingdoms and for diverse types of cell death.     

Autophagic cell death: Loch Ness monster or endangered species?

The concept of autophagic cell death was first established based on observations of increased autophagic markers in dying cells. The major limitation of such a morphology-based definition of autophagic cell death is that it fails to establish the functional role of autophagy in the cell death process, and thus contributes to the confusion in the literature regarding the role of autophagy in cell death and cell survival. Here we propose to define autophagic cell death as a modality of non-apoptotic or necrotic programmed cell death in which autophagy serves as a cell death mechanism, upon meeting the following set of criteria: (i) cell death occurs without the involvement of apoptosis; (ii) there is an increase of autophagic flux, and not just an increase of the autophagic markers, in the dying cells; and (iii) suppression of autophagy via both pharmacological inhibitors and genetic approaches is able to rescue or prevent cell death. In light of this new definition, we will discuss some of the common problems and difficulties in the study of autophagic cell death and also revisit some well-reported cases of autophagic cell death, aiming to achieve a better understanding of whether autophagy is a real killer, an accomplice or just an innocent bystander in the course of cell death. At present, the physiological relevance of autophagic cell death is mainly observed in lower eukaryotes and invertebrates such as Dictyostelium discoideum and Drosophila melanogaster. We believe that such a clear definition of autophagic cell death will help us study and understand the physiological or pathological relevance of autophagic cell death in mammals.     

Autophagic cell death: Loch Ness monster or endangered species?

The concept of autophagic cell death was first established based on observations of increased autophagic markers in dying cells. The major limitation of such a morphology-based definition of autophagic cell death is that it fails to establish the functional role of autophagy in the cell death process, and thus contributes to the confusion in the literature regarding the role of autophagy in cell death and cell survival. Here we propose to define autophagic cell death as a modality of non-apoptotic or necrotic programmed cell death in which autophagy serves as a cell death mechanism, upon meeting the following set of criteria: (i) cell death occurs without the involvement of apoptosis; (ii) there is an increase of autophagic flux, and not just an increase of the autophagic markers, in the dying cells; and (iii) suppression of autophagy via both pharmacological inhibitors and genetic approaches is able to rescue or prevent cell death. In light of this new definition, we will discuss some of the common problems and difficulties in the study of autophagic cell death and also revisit some well-reported cases of autophagic cell death, aiming to achieve a better understanding of whether autophagy is a real killer, an accomplice or just an innocent bystander in the course of cell death. At present, the physiological relevance of autophagic cell death is mainly observed in lower eukaryotes and invertebrates

such as Dictyostelium discoideum and Drosophila melanogaster. We believe that such a clear definition of autophagic cell death will help us study and understand the physiological or pathological relevance of autophagic cell death in mammals.     

Cleavage of Atg3 protein by caspase-8 regulates autophagy during receptor-activated cell death

Autophagy is an evolutionarily conserved mechanism contributing to cell survival under stress conditions including nutrient and growth factor deprivation. Connections and cross-talk between cell death mechanisms and autophagy is under investigation. Here, we describe Atg3, an essential regulatory component of autophagosome biogenesis, as a new substrate of caspase-8 during receptor-mediated cell death. Both, tumor necrosis factor α and tumor necrosis factor-related apoptosis inducing ligand induced cell death was accompanied by Atg3 cleavage and this event was inhibited by a pan-caspase inhibitor (zVAD) or a caspase-8-specific inhibitor (zIETD). Indeed, caspase-8 overexpression led to Atg3 degradation and this event depended on caspase-8 enzymatic activity. Mutation of the caspase-8 cleavage site on Atg3 abolished its cleavage both in vitro and in vivo, demonstrating that Atg3 was a direct target of caspase-8. Autophagy was inactive during apoptosis and blockage of caspases or overexpression of a non-cleavable Atg3 protein reestablished autophagic activity upon death receptor stimulation. In this system, autophagy was important for cell survival since inhibition of autophagy increased cell death. Therefore, Atg3 provides a novel link between apoptosis and autophagy during receptor-activated cell death.     

Caspase-8 activation and bid cleavage contribute to MCF7 cellular execution in a caspase-3-dependent manner during staurosporine-mediated apoptosis

There are at least two distinct classes of caspases, initiators (e.g. caspases-8, -9, and -10) and effectors (e.g. caspase-3). Furthermore, it is believed that there are two distinct primary apoptotic signaling pathways, one of which is mediated by death receptors controlled by caspases-8/10, and the other by the release of cytochrome c and activation of a caspase-9/Apaf1/cytochrome c apoptosome. However, several recent reports have demonstrated that caspase-8, and its substrate Bid, are frequently activated in response to certain apoptotic stimuli in a death receptor-independent manner. These results suggest that significant cross-talk may exist between these two distinct signaling arms, allowing each to take advantage of elements unique to the other. Here we provide evidence that activation of caspase-8, and subsequent Bid cleavage, does indeed participate in cytochrome c-mediated apoptosis, at least in certain circumstances and cell types. Furthermore, the participation of activated caspase-3 is essential for activation of caspase-8 and Bid processing to occur. Although caspase-8 activation is not required for the execution of a cytochrome c-mediated death signal, we found that it greatly shortens the execution time. Thus, caspase-8 involvement in cytochrome c-mediated cell death may help to amplify weaker death signals and ensure that apoptosis occurs within a certain time frame.     

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.