7Autophagy – The Liaison between the Lysosomal System and Cell Death

1. INTRODUCTION

The involvement of lysosomes, the organelle with the highest concentration of hydrolases, in cellular death has been extensively analyzed in different contexts in the past. In many of those studies, lysosomes were proposed to play a “passive” role in the cellular death process, resulting from the leakage of potent lysosomal enzymes into the cytosol. In fact, rupture of the lysosomal membrane after various types of cellular injury or under certain pathological conditions can lead to both apoptotic and nonapoptotic cell death. For example, lysomotropic agents, certain lipid products such as sphingosine or ceramide, a wide variety of death stimuli such as death receptor activation, p53 activation, microtubule-stabilizing agents, oxidative stress, and growth factor deprivation induce lysosomal permeabilization and the release of lysosomal proteases, generically known as cathepsins, into the cytosol. Studies using both genetic and pharmacological blockage of cathepsins support that cytosolic release of these lysosomal hydrolases can mediate caspase-dependent and -independent cell death.

The involvement of lysosomes in cellular death has recently been revisited, and a more active role for this organelle has been proposed. The main drive for this re-evaluation has been the recent advances in our understanding of one of the basic lysosomal functions: autophagy, or the self-digestion of intracellular components by lysosomes. In contrast to the “passive” role in cell death in which lysosomes are merely the carriers of the damaging enzymes that are released into the cytosol, in recent years a direct contribution of lysosomes as intact and properly functioning organelles to cell death has been proposed. The concept of autophagic cell death, however, is not new for cell death researchers.

In fact, this term has been extensively used in the classification of programmed cell death types based on morphological features (Figure 7-1). Thus, whereas in cell death type I or apoptotic cell death, the signature features are condensation of the nucleus and cytoplasm, DNA fragmentation, and early collapse of cytoskeletal elements, but preservation of organelles until late stages, the term cell death type II, or autophagic cell death, has been reserved for dying cells in which the presence of autophagosomes and autophagolysosomes – vesicular compartments related to autophagy – are the predominant morphological feature. In this case, early degradation of organelles but preservation of cytoskeletal elements until late stages are the signature features.

This connection between autophagy and cell death has come more as a shock to autophagy researchers, because most studies support a role for autophagy in sustaining cell survival. In this chapter we review some of the recent advances in the understanding of autophagy resulting from the better molecular characterization of this pathway and how the ability to genetically and pharmacologically modulate this lysosomal pathway has shed light on this apparent paradox.

2. AUTOPHAGY

Degradation of intracellular components is essential for maintenance of cellular homeostasis, continuous renewal of the proteome and organelles, removal of damaged cellular constituents, and the cellular response to environmental stressors. Two major proteolytic systems participate in intracellular protein clearance: (1) the ubiquitin/proteasome system, and (2) the lysosome. This degradation of intracellular proteins and organelles within lysosomes is called autophagy, a process conserved from yeast to mammalian cells.

Figure 7-4. Physiologic functions of autophagy. Three of the many important functions of autophagy are to (1) become an alternative source of energy when nutrients are scarce by degrading proteins and lipid stores into amino acids (aa) and free fatty acids (FA); (2) contribute to quality control by eliminating any damaged protein and intracellular component; and (3) contribute to cellular defense against extracellular pathogens.

starvation that neonatal tissues face right after birth until nutrients can be restored through nursing.

Activation of autophagy during other types of stress responds, for the most part, to the need to eliminate intracellular components damaged by the different stressors (Figure 7-4). In other instances, activation of autophagy is used by cells as a mechanism of defense against pathogen invasion because autophagosomes have the capability to sequester and deliver the intruder to the lysosomal system for degradation.

Added to these well-characterized functions of autophagy as an inducible or stress-activated process, studies in the last few years have revealed a major role for autophagy in maintenance of cellular homeostasis under basal conditions. This basal autophagy has demonstrated to be an essential component of the cellular quality control mechanisms that prevent accumulation of cytotoxic proteins (aggregated and/or misfolded) and malfunctioning organelles (e.g., depolarized mitochondria, regions of the endoplasmic reticulum undergoing proteotoxic stress, nonfunctional peroxisomes). Autophagic removal of mitochondria is of particular relevance in the context of programmed cell death. Changes in the mitochondrial membrane potential and the resultant cytosolic release of mitochondrial components such as cytochrome c play a central role in the activation/maintenance of different cell death pathways. Consequently, a process such as macroautophagy that is able to sequester and eliminate the faulty

mitochondria could, at least in principle, prevent further engagement of downstream cell death effectors and hence avert cell death. In this respect, major effort is currently being dedicated to elucidate the mechanisms that mediate selective removal of altered mitochondria while preserving functional ones. Although still premature, recent studies support that active mitochondrial fusion and fission allow for the reorganization and sequestration of damaged mitochondrial components into daughter mitochondria that are segregated from the networking pool, recognized by the autophagosome integral components, and eliminated by autophagy.

2.3. Autophagy and human pathology

The improved methods to track autophagic activity in cellular and animal models, along with the capability now to modulate autophagy in vivo through genetic manipulations in ATG genes, has helped establish direct connections between autophagy malfunction and diverse pathologies, including neurodegenerative and metabolic diseases, infectious diseases, cancer, heart disease, and others.

Failure or inadequate autophagy has been proposed to underlie the pathogenesis of many late-onset neurodegenerative disorders caused by intracellular accumulation of pathogenic proteins that organize into toxic oligomers and higher order multimeric structures. As an essential component of the mechanisms for intracellular quality control, autophagy contributes to the continuous removal of these pathogenic proteins preventing cellular toxicity. In fact, upregulation of autophagy in several experimental models of proteotoxicity has proven effective in diminishing accumulation of protein aggregates and slowing down progression of disease. Defects in autophagy, often aggravated with age, have been extensively reported in the affected neurons in many of these disorders, suggesting that failure of this defensive mechanism is behind cellular loss and the subsequent onset of symptoms. Different components of the autophagic system seem to be direct targets of the pathogenic proteins. For example, mutations or post-translational modifications in α-synuclein, the protein that accumulates in the affected neurons in patients with Parkinson’s disease, have been shown to directly interact with the CMA receptor at the lysosomal membrane, resulting in general CMA inhibition. Impaired macroautophagy has been reported in Huntington’s and Alzheimer’s diseases, although the mechanisms behind the autophagic failure remain, for the most part, unknown. In light of these