3. AUTOPHAGY AND CELL DEATH

As described in the introduction, the involvement of autophagy in programmed cell death has been controversial. As expected from a stress-adaptation pathway that should promote cell survival, there is profuse evidence that disruption of autophagy or of the lysosomal system promotes cell death. Evidence in support of this prosurvival role of autophagy is discussed in the first part of this section (Figure 7-5, left). However, recent studies have also proposed that excessive or deregulated autophagy can lead to both apoptotic and nonapoptotic cellular death. This process is different from the described activation of apoptosis due to lysosomal enzyme leakage, which can initiate mitochondrial permeabilization and caspase activation. In the second part of this section, we discuss the arguments in sup-

Abundant evidence supports a cytoprotective function for autophagy in very diverse cellular settings and conditions (summarized in Table 7-1). As described in the previous section, genetic blockage of autophagy by deletion of essential autophagic genes in specific tissues in the mouse causes accumulation of polyubiquitylated protein aggregates, major alterations in cellular organelles, and cellular degeneration, thus arguing that a constitutive, low level of basal autophagy in normal tissues has an essential housekeeping function.

The prosurvival effect of autophagy encompasses the two major functions of this pathway, that of acting as an alternative source of energy and as a means for removal of altered cellular components (Figure 7-4). The

capability of autophagy to maintain a positive cellular energy balance is particularly important during nutrient deficiency. Degradation of proteins and even intracellular lipid storages by autophagosomes generates amino acids and free fatty acids that can be used for de novo protein synthesis to support other metabolic pathways such as tricarboxylic acid cycle or to fuel mitochondrial adenosine triphosphate energy production through β-oxidation. This function of autophagy in recycling underlies the ability of this pathway to sustain life during starvation.

This role of autophagy in maintaining cellular bioenergetics has recently proven essential in conditions other than starvation. Thus autophagy is also activated in response to growth factor deprivation or during hypoxia. The rapid degradation of the glucose transporter that follows growth factor withdrawal leaves cells in a compromised energetic balance, but this is prevented by activation of autophagy, which can maintain intracellular ATP levels compatible with cell survival for several weeks.

As described in the previous section, the ability of autophagy to remove defective intracellular components also has a protective effect against cell death. Removal of toxic forms of proteins by autophagy is essential for neuronal survival in various neurodegenerative disorders. Regarding organelles, mitochondria have been the organelle most extensively analyzed given their critical role in cell death pathway. Both the cell death observed on blockage of basal or inducible autophagy depends on mitochondrial outer membrane permeabilization and subsequent caspase activation. However, recent studies support that timely removal of other compromised organelles by autophagy is also essential in preventing cell death. Thus activation of autophagy is often part of the response to endoplasmic reticulum (ER) stress, and failure to activate autophagy under these conditions precipitates cell death both in yeast and in mammalian cells. The high capability of the autophagic systems may be advantageous in certain conditions for the degradation of the compromised ER when compared with the proteasome-mediated degradation of misfolded ER proteins.

Although activation of autophagy has been observed in multiple cellular conditions and in response to numerous stressors, the most convincing evidence of the prosurvival role of this pathway has resulted from genetic studies. Blockage of autophagosome formation in many of those conditions precipitates cell death, supporting thus that the observed activation of autophagy is a cellular survival strategy.

3.2. Autophagy as a cell death mechanism

Autophagic cell death has been historically defined by morphological criteria, namely presence of structures compatible with autophagosomes in a dying cell. However, in recent years it has become clear that the mere presence of autophagosomes is insufficient to distinguish cell death with autophagy from cell death by autophagy. In that respect, the most convincing way to show active participation of autophagy in cell death in a given situation is to demonstrate that blockage of autophagy by manipulation of essential autophagic genes prevents cell death. In fact, multiple reports have now shown decreased apoptosis on inhibition of autophagy under different conditions. For example, silencing of Atg7 or Beclin-1 inhibits the autophagic cell death of L929 cells induced by the pancaspase inhibitor Z-VAD-fmk (N-benzyloxycarbonyl- Val-Ala-Asp- fluoromethylketone). Similarly, embryonic fibroblasts derived from mice that lack the function of the Bcl-2 family member (Bax−/–Bak−/– MEF) are resistant to apoptosis (by treatment with etoposide) but die by autophagic cell death that requires Atg5 and Atg6 function.

However, as a note of caution, a beneficial prosurvival effect of blockage of autophagy can be misleading under certain conditions. Thus, for example, during autophagic stress resulting from the inability of lysosomes to clear autophagosomes, a decrease in autophagosome formation may give the cells a temporary “break.” Massive accumulation of autophagosomes inside cells, as the one observed in some neurodegenerative disorders or in some vacuolar myopathies, results in major alterations in cellular trafficking, energetic dysbalance, and often compromised stability of the autophagosome, with the consequent cytosolic leakage of undegraded toxic cellular products. Under these conditions, knockdown of the genes involved in autophagosome formation will reduce the total autophagosome content and may give time to the lysosomal system to accommodate the clearance of the remaining autophagosomes, with the consequent beneficial effect for the cell. Often, in these circumstances, the original upregulation of the autophagic system was part of the cellular defense against intraor extracellular stressors, and consequently, it should not be classified as cellular death by autophagy, because it is the failure to perform a complete degradative autophagy that leads to compromised cell viability. Different authors have proposed the more appropriate term cell death with autophagy to refer to these conditions. One additional variation on this theme has been