Autophagy as a trigger for cell death

Abstract

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.

Dying cells often display features of autophagy, such as autophagic vacuoles varying in morphology, number and content. However, it is not clear if autophagy causes cell death, acts in parallel to cell death or just clears up the dead corpses where macrophages are not able to implement their task. A growing body of evidence suggests that autophagy acts upstream of, and/or in parallel to, cell death during Drosophila development. In particular, autophagy promotes caspase-dependent cell death in the amnioserosa during Drosophila embryogenesis. atg1 overexpression in the larval fat body induces autophagy and cell death in a caspase- dependent manner. In larval salivary gland cells, genetic inhibition of atg genes leads to incomplete gland degeneration, and the combined inhibition of autophagy and caspases enhances the incomplete destruction of salivary glands. Likewise, mutation of atg7 results in an inhibition of DNA fragmentation during early oogenesis and in the midgut. In addition, degradation of the larval midgut appears to depend on autophagy, but not caspases. Despite this accumulating evidence, the mechanism by which autophagy can promote cell death is not clear.

In our recent study we set out to determine the role of autophagy in developmental cell death during late oogenesis in Drosophila, and how autophagy can act as a pro-death factor. Oogenesis in Drosophila is a well-established model to study cell death. Developmental cell death of the nurse cells occurs during late stages of oogenesis and is required for the normal maturation of the egg chamber. To address the role of autophagy in nurse cell death we generated a novel autophagy reporter, the EGFP-mCherry-DrAtg8a transgene. This reporter appears yellow (green merged with red) in nonacidic autophagosomes, and is red only in the acidic autolysosomes due to quenching of the GFP. When we expressed this reporter in the nurse cells we observed several yellow puncta in the cytoplasm of nurse cells during developmental stage 12, which became red during developmental stage 13 when the majority of nurse cells are degenerated. Atg8a puncta were located either adjacent to or attached to the fragmented nurse cell nucleus, indicative of nuclear autophagy. To examine the role of autophagy in nurse cell death, we generated germline mutant cells for the Drosophila autophagy genes atg1, atg13 and vps34, and examined them for cell death using the TUNEL assay in order to detect DNA fragmentation. We observed that in all of the autophagy mutants tested, there was a significant increase in the number of stage 14 egg chambers with persisting TUNEL-negative nurse cell nuclei. This phenotype differs from wild-type stage 14 egg chambers where nurse cell nuclei can rarely be detected, and those few that remain are exclusively TUNEL-positive. Autophagy germline mutants also exhibit attenuation of cleaved caspase-3 expression compared to the wild type. Together these data demonstrate that autophagy is required for nurse cell death and degradation during late oogenesis in D. melanogaster, and that autophagy functions upstream of caspase- 3 processing and DNA fragmentation in this developmental context.

Our hypothesis was that proteins crucial for cell survival could be degraded by autophagy, thus promoting cell death. To test this we investigated the localization of Drosophila inhibitors of apoptosis in the nurse cells during late oogenesis, and their relationship to the autophagic marker GFP-Atg8a. We found that one of these apoptosis inhibitors, dBruce, colocalizes with the autophagosome marker GFP-Atg8a during developmental stages 12 and 13. Furthermore, dBruce accumulates in nurse cell cytoplasm of atg1, atg13 and vps34 germline mutants. In addition, double-mutant egg chambers for either atg1 and dBruce or vps34 and dBruce contain persistent nurse cell nuclei that are TUNEL-positive. These data indicate that dBruce is required for nurse cell survival during oogenesis by controlling DNA fragmentation.

dBruce belongs to the inhibitor of apoptosis protein family. It contains both BIR (Baculoviral IAP Repeat—responsible for caspase inhibition) and UBC (UBiquitin Conjugation—responsible for ubiquitin conjugation) domains in N-and C-termini, respectively. This giant protein could potentially inhibit caspases and consequently cell death, by two potential mechanisms. First, this could occur by binding to caspases via the BIR domain and subsequently inhibiting their activity, as has been shown for other IAP family members. Alternatively, dBruce could assist ubiquitination of caspases by acting as an E2 ubiquitin-conjugating enzyme. Interestingly, inactivation of Drosophila effector caspase drICE through nondegradative polyubiquitination has been recently shown. drICE was found to be active during late Drosophila oogenesis in the nurse cells, and we propose that it may be regulated by dBruce. drICE activates dCAD (Caspase-activated DNase). It was recently shown that dICAD and DNaseII participate in nurse cells death in late Drosophila oogenesis and that dICAD mutants possess a significant decrease in TUNEL-positive staining. Interestingly, dying nurse cells exhibit characteristics of programmed necrosis, and the lysosomal genes dor, spinster and cathepsin D are required for this process. Additionally, atg8a was identified as an enhancer of spinster, further supporting the requirement of autophagy genes for cell death.

dBruce is known to play a crucial role during sperm individualization in Drosophila where caspase activity must be tightly controlled and restricted to a particular region in the cell. During Drosophila oogenesis the nurse cells are sacrificed for their sister cell, the oocyte, after they have fulfilled their role by providing the nutrients necessary for future embryonic development. Nurse cell death must also be very carefully regulated in order to protect the oocyte from lethal caspase activity, and to prevent premature degradation of nurse cells. We suggest that dBruce is a critical regulator of cell death during Drosophila oogenesis, and propose the following model for nurse cell death and degradation during late oogenesis, where apoptosis, autophagy and necrosis participate: At the late stages of oogenesis, the communication of nurse cells with the oocyte is blocked by the complete encapsulation of the oocyte by the centripetal follicle cells. At this point, the nurse cells have already transferred most of their cytoplasm to the oocyte. Autophagy is then activated to degrade dBruce and other cytoplasmic material in the remaining nurse cell cytoplasm. We speculate that activation of autophagy may be controlled by the transcription factor E2F1, which regulates the transcription of the autophagy- related genes LC3, Atg1, Atg5 and DRAM in mammalian cells. Interestingly, mutations in the Drosophila dE2F1 homologue result in abnormal stage 14 egg chambers containing persisting nurse cell nuclei that are not TUNEL-positive. Autophagic degradation of dBruce promotes limited activation of drICE that can lead to dCAD-mediated DNA fragmentation and subsequently DNase II can function to degrade nurse cell DNA further in autolysosomes. In conclusion, our findings indicate that autophagy plays an important role in nurse cell death during late oogenesis in Drosophila, first by acting upstream of DNA fragmentation, thereby causing cell death, and then by scavenging nurse cell remnants.

Nezis IP, Shravage BV, Sagona AP, Lamark T, Bjørkøy G, Johansen T, Rusten TE, Brech A, Baehrecke EH, Stenmark H. Autophagic degradation of dBruce controls DNA fragmentation in nurse cells during late Drosophila melanogaster oogenesis. J Cell Biol. 2010;190:523–31. doi: 10.1083/jcb.201002035.