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Apoptosis is a form of regulated cell death (RCD) that involves proteases of the caspase family. Pharmacological and genetic strategies that experimentally inhibit or delay apoptosis in mammalian systems have elucidated the key contribution of this process not only to (post-)embryonic development and adult tissue homeostasis, but also to the etiology of multiple human disorders. Consistent with this notion, while defects in the molecular machinery for apoptotic cell death impair organismal development and promote oncogenesis, the unwarranted activation of apoptosis promotes cell loss and tissue damage in the context of various neurological, cardiovascular, renal, hepatic, infectious, neoplastic and inflammatory conditions. Here, the Nomenclature Committee on Cell Death (NCCD) gathered to critically summarize an abundant pre-clinical literature mechanistically linking the core apoptotic apparatus to organismal homeostasis in the context of disease., (© 2023. The Author(s), under exclusive licence to ADMC Associazione Differenziamento e Morte Cellulare.)

A major function of ubiquitylation is to deliver target proteins to the proteasome for degradation. In the apoptotic pathway in Drosophila, the inhibitor of apoptosis protein 1 (Diap1) regulates the activity of the initiator caspase Dronc (death regulator Nedd2-like caspase; caspase-9 ortholog) by ubiquitylation, supposedly targeting Dronc for degradation by the proteasome. Using a genetic approach, we show that Dronc protein fails to accumulate in epithelial cells with impaired proteasome function suggesting that it is not degraded by the proteasome, contrary to the expectation. Similarly, decreased autophagy, an alternative catabolic pathway, does not result in increased Dronc protein levels. However, combined impairment of the proteasome and autophagy triggers accumulation of Dronc protein levels suggesting that autophagy compensates for the loss of the proteasome with respect to Dronc turnover. Consistently, we show that loss of the proteasome enhances endogenous autophagy in epithelial cells. We propose that enhanced autophagy degrades Dronc if proteasome function is impaired.

While most programmed cell death (PCD) in animal development is reliant upon the caspase-dependent apoptotic pathway and subsequent cleavage of caspase substrates, we found that PCD in Drosophila larval midgut occurs normally in the absence of the main components of the apoptotic machinery. However, when some of the components of the autophagic machinery were disrupted, midgut destruction was severely delayed. These studies demonstrate that Drosophila midgut PCD is executed by a novel mechanism where caspases are apparently dispensable, but that requires autophagy.

Background: Cell growth arrest and autophagy are required for autophagic cell death in Drosophila. Maintenance of growth by expression of either activated Ras, Dp110, or Akt is sufficient to inhibit autophagy and cell death in Drosophila salivary glands, but the mechanism that controls growth arrest is unknown. Although the Warts (Wts) tumor suppressor is a critical regulator of tissue growth in animals, it is not clear how this signaling pathway controls cell growth., Results: Here, we show that genes in the Wts pathway are required for salivary gland degradation and that wts mutants have defects in cell growth arrest, caspase activity, and autophagy. Expression of Atg1, a regulator of autophagy, in salivary glands is sufficient to rescue wts mutant salivary gland destruction. Surprisingly, expression of Yorkie (Yki) and Scalloped (Sd) in salivary glands fails to phenocopy wts mutants. By contrast, misexpression of the Yki target bantam was able to inhibit salivary gland cell death, even though mutations in bantam fail to suppress the wts mutant salivary gland-persistence phenotype. Significantly, wts mutant salivary glands possess altered phosphoinositide signaling, and decreased function of the class I PI3K-pathway genes chico and TOR suppressed wts defects in cell death., Conclusions: Although we have previously shown that salivary gland degradation requires genes in the Wts pathway, this study provides the first evidence that Wts influences autophagy. Our data indicate that the Wts-pathway components Yki, Sd, and bantam fail to function in salivary glands and that Wts regulates salivary gland cell death in a PI3K-dependent manner.

Caspase family proteases play important roles in the regulation of apoptotic cell death. Initiator caspases are activated in response to death stimuli, and they transduce and amplify these signals by cleaving and thereby activating effector caspases. In Drosophila, the initiator caspase Nc (previously Dronc) cleaves and activates two short-prodomain caspases, Dcp-1 and Ice (previously Drice), suggesting these as candidate effectors of Nc killing activity. dcp-1-null mutants are healthy and possess few defects in normally occurring cell death. To explore roles for Ice in cell death, we generated and characterized an Ice null mutant. Animals lacking Ice show a number of defects in cell death, including those that occur during embryonic development, as well as during formation of adult eyes, arista and wings. Ice mutants exhibit subtle defects in the destruction of larval tissues, and do not prevent destruction of salivary glands during metamorphosis. Cells from Ice animals are also markedly resistant to several stresses, including X-irradiation and inhibition of protein synthesis. Mutations in Ice also suppress cell death that is induced by expression of Rpr, Wrinkled (previously Hid) and Grim. These observations demonstrate that Ice plays an important non-redundant role as a cell death effector. Finally, we demonstrate that Ice participates in, but is not absolutely required for, the non-apoptotic process of spermatid differentiation.

Autophagy is used to degrade components of the cytoplasm and functions as a cell survival mechanism during nutrient deprivation. Autophagic structures have also been observed in many types of dying cells, but experimental evidence for autophagy playing a role in the regulation of programmed cell death is limited. We have recently shown that the autophagy genes Atg7 and Beclin1 are required for the death of certain cells, thus demonstrating that this mechanism of proteolysis is involved in both survival and death. The factors that enable autophagy to regulate distinct cell survival and death responses are not clear, and future work is needed to determine the mechanism(s) that regulate autophagic cell death.

Caspases play a central role in apoptosis, a well-studied pathway of programmed cell death. Other programs of death potentially involving necrosis and autophagy may exist, but their relation to apoptosis and mechanisms of regulation remains unclear. We define a new molecular pathway in which activation of the receptor-interacting protein (a serine-threonine kinase) and Jun amino-terminal kinase induced cell death with the morphology of autophagy. Autophagic death required the genes ATG7 and beclin 1 and was induced by caspase-8 inhibition. Clinical therapies involving caspase inhibitors may arrest apoptosis but also have the unanticipated effect of promoting autophagic cell death.

Self-digestion of cytoplasmic components is the hallmark of autophagic programmed cell death. This auto-degradation appears to be distinct from what occurs in apoptotic cells that are engulfed and digested by phagocytes. Although much is known about apoptosis, far less is known about the mechanisms that regulate autophagic cell death. Here we show that autophagic cell death is regulated by steroid activation of caspases in Drosophila salivary glands. Salivary glands exhibit some morphological changes that are similar to apoptotic cells, including fragmentation of the cytoplasm, but do not appear to use phagocytes in their degradation. Changes in the levels and localization of filamentous Actin, alpha-Tubulin, alpha-Spectrin and nuclear Lamins precede salivary gland destruction, and coincide with increased levels of active Caspase 3 and a cleaved form of nuclear Lamin. Mutations in the steroid-regulated genes beta FTZ-F1, E93, BR-C and E74A that prevent salivary gland cell death possess altered levels and localization of filamentous Actin, alpha-Tubulin, alpha-Spectrin, nuclear Lamins and active Caspase 3. Inhibition of caspases, by expression of either the caspase inhibitor p35 or a dominant-negative form of the initiator caspase Dronc, is sufficient to inhibit salivary gland cell death, and prevent changes in nuclear Lamins and alpha-Tubulin, but not to prevent the reorganization of filamentous Actin. These studies suggest that aspects of the cytoskeleton may be required for changes in dying salivary glands. Furthermore, caspases are not only used during apoptosis, but also function in the regulation of autophagic cell death.

Cytochrome c is a critical regulator of apoptosome assembly, caspase activation, and programmed cell death. Recent work demonstrates that cytochrome c and caspases function in Drosophila sperm cell differentiation and indicates that caspase activity can be regulated in a subcellular manner in cells that live.

The steroid hormone ecdysone regulates both cell differentiation and cell death during insect metamorphosis, by hierarchical transcriptional regulation of a number of genes, including the Broad-Complex (BR-C), the zinc finger family of transcription factors. These genes in turn regulate the transcription of a number of downstream genes. DRONC, a key apical caspase in Drosophila, is the only known caspase that is transcriptionally regulated by ecdysone during development. We demonstrate that dronc gene expression is ablated or reduced in BR-C mutant flies. Using RNA interference in an ecdysone-responsive Drosophila cell line, we show that DRONC is essential for ecdysone-mediated cell death, and that dronc upregulation in these cells is controlled by BR-C. Finally, we show that the dronc promoter has BR-C interaction sites, and that it can be transactivated by a specific isoform of BR-C. These results indicate that BR-C plays a key role in ecdysone-mediated caspase regulation.