Autophagic cell death: characteristics and regulation

Autophagy, a lysosomal degradation pathway, is essential for cell survival and homeostasis, and prevents numerous pathologies, including infections, cancer, neurodegeneration, aging and heart disease. Though most of the current data suggest that autophagy is cytoprotective, its self-cannibalistic nature has been reported to be deleterious to cell survival. In fact, autophagy has been described as a form of programmed cell death (so-called autophagic cell death or PCD Type II). However, dying cells with autophagic vacuolization may indicate death in spite of, rather than by autophagy. Autophagy may have been activated as a failed attempt at homeostatic self defense. Speculation aside, both the validity and potential mechanisms of autophagic cell death clearly remain enigmatic, despite the importance of this process in medicine and disease prevention. A major problem in monitoring autophagic death in mammalian cells is that this type of cell death lacks, by definition, apoptotic or necrotic markers, and cannot be readily distinguished by any of the commonly used cell death assays in higher organisms. Yeast allows easy detection of cell death by clonogenic assays and has been used extensively to study the fields of both cell death and autophagy independently. Herein, we propose the use of yeast to follow preliminary findings that are strongly suggestive of a bona fide autophagic cell death subroutine. Our preliminary data indicate that ER stress (elicited by tunicamycin) leads to cell death in yeast which can be rescued by deletion of components of the autophagic machinery (ATG5 and ATG7). We will initially test whether signalling components of the ER stress response (UPR), such as IRE1, HAC1, or their downstream targets (like XBP1) are necessary or sufficient to trigger a form of cell death that lacks conventional markers, but displays features of autophagy. We predict this death also to be rescued by deletion of the autophagic machinery. To establish a hierarchy of the events we observed, we will use epistasis experiments: we will overexpress ATG genes in the background of UPR mutants, and vice versa, and determine cell death. We also found that depletion of methionine (but not of other amino acids), leads to cell death that is accompanied by autophagic markers (yet devoid of conventional death markers) and abrogated by deletion of autophagic genes. Methionine depletion likely also triggers ER stress due to incomplete translation and protein aggregation. We will delineate putative up- and down- stream mechanisms regulating autophagic death upon methionine depletion. We will determine, for instance, whether components of the ER stress pathway, the TOR pathway or the ATG gene products themselves are necessary for autophagic death upon methionine depletion. In addition, a preliminary screen revealed that the protein phosphatase 2A (PP2A) (also linked to the TOR pathway) is a candidate regulator of autophagic cell death. Finally, we will use both our established scenarios of autophagic cell death for proteomic and metabolomic analysis. These efforts are expected to lead to the first molecular description of this poorly understood, yet critically important, cell death modality.

During cellular life many challenging situations must be overcome. One of the major mechanisms to support a long and healthy lifespan is called autophagy (from the Greek self-eating). It is a cellular self-recycling process, in which damaged or unneeded cellular material (from single proteins to whole-cell compartments) can be removed without posing further threat to the cells survival. Even if nowadays autophagy is mainly seen as a pro-survival mechanism, it was originally described as a form of cell death following the observation that increased autophagy levels are frequently observed during various cell death processes. However, this observation might be the result of a homeostatic counter-reaction of the cell against a deadly insult, or in other words: in many of the reported conditions, cells are likely to have died in spite of and not by autophagy. We wanted to improve our knowledge on the duality of this process and define if besides its pro-survival effects - there is a lethal side to it. We investigated cells from nonadipose tissues challenged with high doses of palmitic acid, which leads to lipotoxicity (toxic fat) und eventually cell death. We found that palmitic acid activates autophagy and - instead of counteracting cell death - promotes it. In turn, the inhibition of autophagy could prevent death, speaking for an active role of autophagy in this specific lethal process. Furthermore, we could delineate two metabolites that are key in the regulation of age-induced cell death. The first one is the naturally occurring amino acid methionine, an essential food component. We found that moderate restriction of the availability of methionine in a yeast aging model extends chronological lifespan by approximately 30 to 40%. This pro-survival effect clearly depends on autophagy, which is induced by methionine restriction and enhances the function of the lysosome, the cellular compartment that processes molecular waste. The second metabolite is Acetyl-CoA, a molecular energy sensor that delivers information about food availability. We could show that high production of this sensor leads to reduced removal of unwanted cellular material whereas low levels induce autophagy, promoting longevity in flies and yeast. Thus, both situations (methionine and Acetyl-CoA limitation) demonstrate a pro-survival function of autophagy that improves survival fitness. In the course of this project, the lethal and protective aspects of autophagy were investigated and our results led to significant improvements in the understanding of this delicate balance and its regulation.