MITOCH-00937; No of Pages 9 Mitochondrion xxx (2014) xxx–xxx

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Mitochondrion journal homepage: www.elsevier.com/locate/mito

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Urška Repnik a,b, Maruša HafnerČesen a, Boris Turk a,c,d,⁎ a

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Late endocytic compartments include late endosomes, lysosomes and hybrid organelles. In the acidic lumen, cargo material derived from endocytosed and phagocytosed extracellular material and autophagyderived intracellular material is degraded. In the event of lysosomal membrane permeabilization (LMP), the function of endo/lysosomal compartment is affected and the luminal contents are released into the cytosol to various extents. LMP can be a result of osmotic lysis or direct membranolytic activity of the compounds that accumulate in the lumen of endo/lysosomes. In addition to several synthetic compounds, such as dipeptide methyl esters and lysosomotropic detergents, endogenous agents that can cause LMP include ROS and lipid metabolites such as sphingosine and phosphatidic acid. Depending on the cell type and the dose, LMP can initiate the lysosomal apoptotic pathway, pyroptosis or necrosis. LMP can also amplify cell death signaling that was initiated outside the endocytic compartment, and hamper cell recovery via autophagy. However, mechanisms that connect LMP with cell death signaling are poorly understood, with the exception of the proteolytic activation of Bid by aspartic cathepsin D and cysteine cathepsins. Determination of LMP in a cell model system is methodologically challenging. Even more difficult is to prove that LMP is the primary event leading to cell death. Nevertheless, LMP may prove to be a valuable approach in therapy, either as a trigger of cell death or as a mechanism of therapeutic drug release in the case of delivery systems that target the endocytic pathway. © 2014 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

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Keywords: Lysosomal membrane permeabilization Lysosomes Cathepsins Cell death

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1. Introduction

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The endocytic pathway is a system of organelles that is specialized for the uptake, sorting and degradation of the extracellular material as well as degradation of intracellular cytoplasmic material delivered by autophagy. The early endocytic pathway is represented by endocytic vesicles, which arise at the plasma membrane, and early endosomes, whereas the late endocytic pathway consists of late endosomes, lysosomes and hybrid organelles. The two parts are connected by multivesicular bodies (MVB), alternatively called endosomal carrier vesicles (ECV) (Griffiths, 1996). In the context of cell death the term lysosomes usually represents late endocytic organelles (Repnik et al., 2013). Integral membrane proteins in endosomes and lysosomes are involved in membrane trafficking and transport of small metabolites across the limiting membrane. In addition, late endocytic organelles

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⁎ Corresponding author at: Faculty of Chemistry and Chemical Technology, University of Ljubljana, Aškerčeva 5, 1000 Ljubljana, Slovenia. Tel.: +386 1 477 3772; fax: +386 1 477 3984. E-mail address: [email protected] (B. Turk).

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Department of Biochemistry and Molecular and Structural Biology, Jožef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia Department of Biosciences, University of Oslo, Blindernveien 31, 0371 Oslo, Norway Center of Excellence CIPKeBiP, Jamova 39, 1000 Ljubljana, Slovenia d Faculty of Chemistry and Chemical Technology, University of Ljubljana, Aškerčeva 5, 1000 Ljubljana, Slovenia b

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Lysosomal membrane permeabilization in cell death: Concepts and challenges

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collectively contain over 50 soluble hydrolytic enzymes, which are involved in the degradation of the luminal cargo (Schroder et al., 2010). Among them are proteases, including the aspartic cathepsin D and cysteine cathepsins B, C, H, K, L, S and X (Turk and Turk, 2009; Turk et al., 2012b). The other major hydrolases are various lipases such as acid sphingomyelinase, acid ceramidase, acid lipase and acid phospholipase A2, and glycosidases responsible for the degradation of oligosaccharides, polysaccharides and removal of carbohydrate moieties from glycolipids and glycopeptides (Schroder et al., 2010). One of the major characteristics of the endocytic pathway is its progressive decrease in pH, which assists cargo degradation. Lysosomal proteases have adapted to the acidic environment by their structural stability. At neutral pH, some, like cathepsin S, can remain active for several hours, others, such as cathepsin D, will lose activity due to de-protonation of the residues in the active site, but remain structurally stable, and still others, such as cathepsin L, will unfold (Turk and Turk, 2009). Either way, activity preserved even for a short period of time is postulated to be sufficient for proteolytic modification of molecules involved in cell death signaling (Turk et al., 2012a). The risk of the release of lysosomal hydrolases into the cytosol for cell survival has been recognized already by de Duve (de Duve, 1959).

http://dx.doi.org/10.1016/j.mito.2014.06.006 1567-7249/© 2014 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

Please cite this article as: Repnik, U., et al., Lysosomal membrane permeabilization in cell death: Concepts and challenges, Mitochondrion (2014), http://dx.doi.org/10.1016/j.mito.2014.06.006

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40 years ago, esters of amino acids at a concentration of 0.5–20 μM were found to rupture isolated rat lysosomes (Goldman and Kaplan, 1973; Reeves, 1979). The pKa of the amino group in amino acid methyl esters is around 7.7 and so which is considerably lower compared to free amino acids, in which it is usually above 9 (Jencks and Regenstein, 1970). Because of the relatively low pKa, a considerable fraction of amino acid methyl ester molecules is not protonated at cytosolic pH and these can permeate freely across membranes. However, it was observed that the extent of the accumulation of amino acids and thereby their potency for the lysosome rupture strongly correlated with the enzymatic reactions that hydrolyzed esters to free amino acids and produced dipeptides in the lysosomal lumen. In addition to low accumulation of D-stereoisomers, in particular accumulation of alanine and serine was smaller compared to phenylalanine and leucine (Goldman and Kaplan, 1973; Reeves, 1979). It was later established that DPPI, also known as cathepsin C, has the major role in this process, and pre-incubation of cells with the cathepsin C inhibitor Gly-Phe-CNH2 abrogates lysosome destabilization (Thiele and Lipsky, 1990; Uchimoto et al., 1999). Upon incubating the cells with dipeptidyl methyl esters, esterified and non-esterified peptides Leu2, Leu4 and Leu6 were identified in the cell lysate and it was further shown that these cathepsin C-generated polypeptide metabolites exert membranolytic activity on red blood cells (Thiele and Lipsky, 1990). In line with cathepsin C specificity preferences (McDonald et al., 1969; McGuire et al., 1992), only dipeptide esters or amides composed entirely of nonpolar R group L-stereoisomer amino acids were converted to membranolytic products by cathepsin C, but not dipeptide esters containing polar amino acid residue such as Ser-Leu-OMe or Leu-Tyr-OMe (Thiele and Lipsky, 1990). Destabilization of lysosomal membrane by amino acid esters therefore seems to be mediated by a combined effect of osmotic lysis due to the accumulation of free amino acids and of membranolytic activity of oligopeptide metabolites. A similar membrane destabilizing effect was observed for dipeptidyl-β-naphtylamides, such as Gly-Phe-β-naphtylamide, which are also hydrolyzed and polymerized by cathepsin C (Berg et al., 1994; Thiele and Lipsky, 1990). Some lysosomotropic compounds exert a proton sponge effect. This phenomenon arises because compounds become protonated at acidic pH in the lumen and thereby become non-permeable. These non-permeable solutes then build up the osmotic pressure across the lysosomal membrane, which causes the inflow of water (Boussif et al., 1995; Kichler et al., 2001). Although chloroquine and ammonium chloride can cause enlargement of endocytic vesicles by the proton sponge effect, they do not readily cause LMP and do not affect the lysosome membrane integrity (Maclean et al., 2008; Wilson et al., 1987), indicating that proton trapping itself is not sufficient to trigger membrane lysis.

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4. Agents that change membrane permeability for small solutes

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Osmotic lysis can be the result of increased membrane permeability for otherwise non permeable solutes, which implies two possible mechanisms. Neutral molecules cross the membrane only by the solubility– diffusion mechanism due to their high solubility in a hydrocarbon phase. Ions are more likely to permeate through transient water defects in membranes. These represent penetration of water molecules into the hydrophobic core of the membrane and act as mobile free volumes that can carry small molecules and ions across the membrane with or without the formation of discrete pores. The frequency of these transient defects is increased by the incorporation of external perturbants and thermal fluctuations of membrane lipids (Deamer and Bramhall, 1986; Gurtovenko and Vattulainen, 2007; Hu et al., 2007). It has

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The hallmark of lysosomal permeabilization is the loss of membrane integrity, which allows the release of luminal contents into the cytosol. Most common mechanisms of LMP are the osmotic lysis or direct destabilization by surfactant activity. LMP-inducing compounds themselves can act as solutes, which upon accumulation in lysosomes increase the osmotic pressure and directly cause the osmotic lysis. Alternatively, certain agents modify the permeability of the limiting membrane so that it becomes permeable to otherwise non-permeable small metabolites that leads to the increased osmotic pressure in the lumen, which eventually causes osmotic lysis. Certain compounds can act as surfactants and cause direct membrane lysis and the release of the luminal contents into the cytosol. Direct osmotic or direct membrane lysis requires that compounds, which mediate LMP, accumulate in the same late endocytic organelles they destabilize. Lysosomotropic amines are membrane permeable compounds that accumulate in lysosomes because protonation in the acidic lumen makes them impermeable (Miller et al., 1983). Less commonly, otherwise permeable compounds become impermeable by enzymatic modification (Goldman and Kaplan, 1973). Membrane nonpermeable compounds are endocytosed at the plasma membrane and then progressively transported along the endocytic pathway so that they accumulate in its terminal part represented by late endosomes and lysosomes. Therefore the sensitivity of cells to LMP induced by a particular compound can depend on the presence or the abundance of particular enzymes, such as cathepsin C for dipeptidyl esters and napthylamides (Goldman and Kaplan, 1973; Thiele and Lipsky, 1990), and can also be affected by the rate of endocytosis. An important factor that determines the sensitivity of the lysosomal membranes to LMP is also their membrane composition, including sphingolipids and cholesterol levels. Sphingolipids contain saturated fatty acids, which allow tight packing and association with the relatively rigid cholesterol molecules. Relatively high content of sphingolipids and cholesterol makes the plasma membrane more ordered, more rigid and thicker compared to the membranes enriched in glycerophospholipids. Membranes of late endocytic organelles contain less sphingolipids and cholesterol than plasma membrane (Hamer et al., 2012), which may also explain their sensitivity to osmotic lysis underlined by naming lysosomes as intracellular osmometers (Lloyd and Forster, 1986). Not surprisingly, increased cholesterol content was identified as a protective factor against LMP (Appelqvist et al., 2011). In contrast, hydrolysis of sphingomyelin increases the risk for LMP (Contreras et al., 2006; Ullio et al., 2012). Lysosomal integral membrane proteins (LIMPs) and lysosomeassociated membrane proteins (LAMPs), whose carbohydrate parts constitute a glycocalyx that lines the inner leaflet of the lysosomal membrane, protect the limiting membrane from the degradative enzymes (Carlsson et al., 1988). It was shown that oncogene-mediated downregulation of these glycoproteins increases the susceptibility to

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2. Mechanisms of LMP

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LMP (Fehrenbacher et al., 2008). Also a chaperon Hsp70, when associated 136 with lysosomal membranes, can protect from LMP in several ways 137 (Johansson et al., 2010; Nylandsted et al., 2004). 138

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Decades later, synthetic and endogenous agents have been identified that can cause lysosomal membrane permeabilization (LMP) with a wide spectrum of consequences for the membrane trafficking, energy metabolism as well as cell survival. In spite of the awareness of the dynamics in the endocytic pathway and the progress in its mechanistic understanding, the concept of the basic nature of the endocytic pathway remains unsettled (Pryor and Luzio, 2009). The model of ‘pre-existing organelles’ (Griffiths, 1996; Griffiths and Gruenberg, 1991), which cannot arise de novo, and the alternative ‘maturation’ model (Russell et al., 2006) both have arguments and supporters. One can speculate that if endocytic compartments cannot arise de novo their irreversible disruption may have deleterious consequences for the cell survival compared to the ability of endocytic compartment to be remade by the maturation of the endocytic vesicles, which form at the plasma membrane.

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With regard to morphological criteria, cell death can appear apoptotic or necrotic. For apoptosis, characterized by nuclear and cytosol fragmentations into apoptotic bodies, activation of caspases is required. Based on biochemical criteria several cell death modalities were proposed for cell demise with apoptotic and necrotic morphology (Kroemer et al., 2009). Lysosomes cannot rival the importance of mitochondrial and plasma membrane integrity for influencing cell survival. However, the cell cannot survive without a functional endocytic system and the arsenal of hydrolases it harbors represents a threat if released into the cytosol. Along these lines, evidence has been obtained for the involvement of lysosomes in biochemically distinct cell death pathways (Fig. 1). The critical event for the involvement of lysosomes in cell death is LMP. The effector mechanisms, which signal cell death downstream of LMP are poorly understood, with the exception of cathepsins. In most cases LMP amplifies cell death signaling, but it can also initiate it, as shown with compounds that specifically target lysosomes. However, even in the absence of LMP, endocytic organelles that are functionally disabled due to increased pH and/or inactive hydrolases will diminish cell's chances for recovery if exposed to stress. In particular, dysfunctional endocytic pathway will disable the removal of damaged organelles by autophagy (Wang et al., 2008). More than a decade ago, a concept was proposed that a limited damage to lysosomes triggers apoptosis and that an extensive one results in necrosis (Kagedal et al., 2001; Li et al., 2000). Over the years, the concept has gained strength and complexity. There is increasing evidence that the same compound can induce different types of cell deaths depending on the cell type and the extent of the accumulation of the compound in the endocytic vesicles. In general, if LMP occurs it affects majority of the late endocytic organelles, and it is the extent of the limiting membrane destabilization that determines the release of lysosomal contents into the cytosol with regard to its molecular size. Release of small metabolites such as protons and other ions may not have serious consequences compared to the release of lysosomal enzymes. However, little is known about the recovery potential of lysosomes following LMP. Moderate LMP mediated with low concentration of Gly-Phe-β-naphtylamide was found to be transient and reversible (Steinberg et al., 2010). Moreover, it was recently shown that in cells with damaged lysosome, lysosomal biogenesis depends on autophagy (Maejima et al., 2013).

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Apoptotic triggers can originate from the outside or from within the cell; these are referred to as the extrinsic or death receptor pathway and the intrinsic or mitochondrial pathway, respectively. Extrinsic apoptosis is initiated upon binding of TNF, TRAIL or FasL to their cognate receptors on the plasma membrane, which leads to the formation of death inducing signaling complex (DISC), a platform for the initiator caspase-8 activation. The intrinsic pathway is the consequence of non-receptor stimuli that include metabolic stress, ER stress, UV radiation, DNA damage and among others as well as LMP. Intrinsic pathways converge on mitochondria, triggering mitochondrial outer membrane permeabilization (MOMP), which is the critical step of the pathway and leads the release of apoptogenic factors into the cytosol (Taylor et al., 2008). Most of the mechanistic understanding of the so called lysosomal apoptotic pathway, which is initiated by LMP, has been obtained in the studies with the

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Lysosomotropic amines with linear-chain hydrocarbon “tails” of 9–14 carbon atoms were found to act as detergents selectively on lysosomal membranes after they accumulate in lysosomes at concentrations above their threshold surfactant concentration, also called critical micelle concentration (CMC), at which they develop surfactant properties. The sigmoidal dose-response shows a high degree of cooperativity and the CMC for weak detergents is higher than that for strong detergents (Firestone et al., 1979). Compounds, which were reported to induce LMP due to detergent activity include O-methyl-serine dodecylamide hydrochloride (MSDH) (Li et al., 2000), N-dodecylimidazole (Boyer et al., 1993; Miller et al., 1983; Wilson et al., 1987) and exogenously added sphingosine (Kagedal et al., 2001). The main drawback of exogenous lysosomotropic detergents is their inefficient targeting. In order to reach CMC, cells need to be treated with relatively high concentrations of these compounds, which although below CMC can destabilize the plasma membrane and impair its barrier function causing necrotic cell death (Aranzazu Partearroyo et al., 1990; Boyer et al., 1993; Forster et al., 1987; Li et al., 2000). This notion is further supported by the observation that cytotoxicity of MSDH is markedly dependent on the cell concentration, which is also true for two other detergents, Triton X-100 and SDS. The explanation is that as the quantity of cell membranes increases the amount of a detergent acting on the plasma membranes becomes insufficient to exert lytic effects (Boyer et al., 1993). In addition, concerns have been raised about the ability of exogenous ceramides to

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reach intracellular membranes (Zager et al., 2000), which also seems relevant for other hydrophobic carbohydrates. An endogenous candidate for a membranolytic compound is lipofuscin (Schutt et al., 2002), which is generated in lysosomes by oxidative polymerization of protein and lipid residues. It is nondegradable and thus accumulates in long-lived postmitotic cells that cannot dilute it by division (Terman and Brunk, 2004).

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been established that fatty acids can readily partition into membranes (Pjura et al., 1984). Increased cytosolic Ca2 + concentration during apoptosis can increase the activity of enzymes, such as phospholipase A2, sphingomyelinase and phospholipase C, which generate lipid metabolites that affect membrane permeability (Johansson et al., 2010). At concentrations above 10 μM, phosphatidic acid and lysophosphatidylcholine, were shown to increase lysosomal membrane permeability to K+ and H+ ions and thus lysosomal osmotic sensitivity (Hu et al., 2007; Yi et al., 2006). Moreover, phosphatidic acid facilitates the insertion of t-Bid, which can be generated by several proteases (Repnik et al., 2012), deep into lipid bilayers, where it undergoes homo-oligomerization and triggers the formation of highly curved nonbilayer lipid phases, thereby allowing the release of luminal contents into the cytosol (Zhao et al., 2012). Similarly, during TNFmediated apoptosis in hematoma cells acid shingomyelinase and acid ceramidase collectively generate sphingosine, which promotes LMP (Ullio et al., 2012) by increasing membrane permeability (Contreras et al., 2006). Another factor with destabilizing effects on lysosomal membranes is reactive oxygen species (ROS). Their action is mediated primarily by peroxidation of membrane lipids. The introduction of oxidized functional groups in lipid tails leads to their conformational change so that the oxidized tails bend toward the water phase and the oxygen atoms form hydrogen bonds with water and the polar lipid head-group. Consequently, the average area per lipid and correspondingly the membrane bilayer thickness increases, and water defects, which allow the permeation of ions, become more frequent, which collectively increase the membrane permeability (Wong-Ekkabut et al., 2007). Lysosomal membranes are particularly prone to ROS-mediated damage, because of the lysosomal iron content, which can produce intralysosomal ROS in the Fenton reaction (Kurz et al., 2008). Most likely, the initial burst of ROS is generated by destabilized mitochondria (Turrens, 2003), although certain compounds, such as gentamicin, can initiate ROS generation inside lysosomes (Denamur et al., 2011). Taken together, enzymatic or oxidative modifications of membrane lipids affect the membrane permeability, which can lead to LMP. This seems to be the major physiologically relevant route to late onset LMP, which occurs downstream of mitochondria destabilization and caspase activation.

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the apoptosome, a platform for the activation of the initiator caspase-9. Activated caspase-9 then proteolytically activates executioner caspases3, -6 and -7, leading to the execution phase of cell death (Taylor et al., 2008). In addition to Bid cleavage, cysteine cathepsins also degrade guardians of mitochondrial integrity, anti-apoptotic Bcl-2 proteins, thereby promoting Bax and Bak activation. Furthermore, downstream of MOMP, cysteine cathepsins can degrade the caspase inhibitor XIAP, which additionally promotes caspase activation (Droga-Mazovec et al., 2008). In addition, apoptosis initiated outside the endocytic compartment can affect lysosome integrity at some point during the signaling pathway and the effectors released from lysosomes then enhance cell demise. Several mechanisms have been proposed for the induction of LMP upstream of MOMP in TNF-α- or TRAIL-induced apoptosis,

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lysosomotropic dipeptide LeuLeu-methyl ester (LLOMe) (Cirman et al., 2004; Droga-Mazovec et al., 2008; Stoka et al., 2001; Uchimoto et al., 1999). In this model, the molecular mechanism linking LMP to MOMP and caspase activation has been identified (Fig. 2). LMP enables the release of lysosomal hydrolases into the cytosol. Especially important are the aspartic cathepsin D (Appelqvist et al., 2012) and cysteine cathepsins (Cirman et al., 2004; Stoka et al., 2001), which in the cytosol process Bid molecule into the proapoptotic form t-Bid. At this stage the lysosomal apoptotic pathway converges with the common intrinsic apoptotic pathway. The conformational change in t-Bid induces a structural change and oligomerization of proapoptotic Bax and Bak proteins (Moldoveanu et al., 2013) resulting in pore formation in the outer mitochondrial membrane. Through these pores, cytochrome c is released into the cytosol where together with the apoptotic protease-activating factor (Apaf-1) it forms

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Fig. 1. Lysosomal membrane permeabilization (LMP) in cell death. Depending on the cell type, the nature and intensity of the stimulus, LPM can initiate or amplify different types of cell death. Extensive rapid LMP results in non-programmed (accidental) necrosis. Crystalline materials can lead to pyroptosis by NLRP3 inflammasome activation. LMP can initiate lysosomal apoptotic pathway by cathepsin-mediated cleavage of Bid and antiapoptotic Bcl-2 homologues, which results in mitochondrial outer membrane permeabilization (MOMP). This pathway is initiated with compounds that directly target lysosomes, such as lysosomotropic detergents. Early LMP has also been implicated in receptor-mediated apoptosis, although signaling downstream of LMP is not well understood. LMP also amplifies signaling at later stages of apoptosis and occurs due to mitochondrial ROS production or activation of lipases.

Fig. 2. Lysosomal apoptotic pathway. LMP, which occurs due to osmotic or direct membrane lysis of late endosomes and lysosomes, results in the release of cathepsins into the cytosol. There, cathepsins process Bid to a pro-apoptotic t-Bid fragment, which assists Bax and Bak oligomerization and thereby the mitochondrial outer membrane permeabilization, which eventually results in caspase activation. Cathepsins additionally promote apoptosis by degradation of anti-apoptotic Bcl-2 homologues and the caspase inhibitor XIAP.

Please cite this article as: Repnik, U., et al., Lysosomal membrane permeabilization in cell death: Concepts and challenges, Mitochondrion (2014), http://dx.doi.org/10.1016/j.mito.2014.06.006

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Macrophages can display a particular type of cell death termed pyroptosis, which is pro-inflammatory. The outcome is a rapid rupture of the plasma membrane with a concomitant release of inflammatory intracellular contents, including IL-1β and IL-18. Characteristic for pyroptosis is the activation of inflammatory capsase-1, which also mediates cell demise. Caspase-1 activation depends on Nod-like receptors (NLRs), which do not activate caspase-1 directly but rather organize a multi-molecular platform termed the inflammasome, which recruits and activates caspase-1. NLR protein 3 (NLRP3) responds to various stimuli including viral DNA and RNA and crystalline material. NLR family CARD domain-containing protein 4 (NLRC4), also known as IPAF, responds to bacterial components and induces pyroptosis of cells infected with bacteria (Bergsbaken et al., 2009). In macrophages, LMP induced with various crystalline materials, such as silica or aluminum salt (alum) crystals, or chemical compounds, such as dipeptidyl methyl ester LeuLeuOMe, can activate NLRP3, which in turn activates caspase-1 and leads to pyroptosis. It appears that lysosomal rupture alone is sufficient to activate the inflammasome without the presence of known inflammasome activators. Bafilomycin A prevents the activation of caspase-1, which indicates the importance

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Until a few years ago the term ‘programmed cell death’ was used only with reference to the apoptotic cell death, whereas necrosis was believed to be non-programmed or ‘accidental’ cell death in response to physico-chemical stress (Leist and Jaattela, 2001). Now it is clear that also necrosis can be driven by well-regulated molecular mechanisms (Galluzzi et al., 2012) and regulated necrosis might be even more prominent than (regulated) apoptosis (Vanden Berghe et al., 2014). All forms of necrosis manifest oncosis, dilated organelles, loss of plasma membrane integrity and the absence of caspase activation and chromatin condensation (Vanden Berghe et al., 2010; Vanlangenakker et al., 2008). The cellular disintegration phase is characterized by an identical sequence of subcellular events, including mitochondrial, lysosomal and plasma membrane permeabilization, although with different kinetics. A detailed comparative study of TNFα-induced necroptosis and H2O2-induced accidental necrosis revealed that lysosomal disruption is a late event coinciding with cellular disintegration phase in necroptosis, but an early event in accidental necrosis (Vanden Berghe et al., 2010). In several reports, LMP-dependent cell death was described as caspase-independent, which is a rather vague definition. If caspases are activated, but cell death cannot be prevented with caspase inhibitors (Mediavilla-Varela et al., 2009; Rammer et al., 2010) cell death can still be classified biochemically as apoptosis. In the intrinsic apoptotic pathway, cells do not die because of activated caspases, but because of metabolic imbalance due to damaged mitochondria, which also activates caspases. In this case, caspase inhibitors can only delay, but not prevent cell death (Kroemer et al., 2009). If caspase activation is absent, then cell death is indeed non-apoptotic and in most cases it fits the description of accidental necrosis (Hornick et al., 2012; Mena et al., 2012; Villalpando Rodriguez and Torriglia, 2013). As already mentioned, lysosomotropic detergents induce apoptosis at concentrations that cause moderate LMP and necrosis at concentrations that cause extensive LMP (Kagedal et al., 2001; Li et al., 2000), although cell death at high concentrations may no longer be LMP-dependent (Boyer et al., 1993; Forster et al., 1987). We have recently reported that depending on the cell type, cells treated with a broad concentration range of the piperidine analog siramesine display either necrotic morphology and die without significant caspase activation, or follow the classic apoptotic scenario (Hafner Česen et al., 2013). In contrast to earlier reports, which argued that siramesine is a lysosomotropic detergent (Ostenfeld et al., 2005, 2008), we demonstrated that lysosomes were alkalinized shortly after the treatment, but not permeabilized. Moreover, LMP, which clearly occurred downstream of MOMP both in apoptotic and necrotic cell deaths, was not a critical event in the cell death signaling (Hafner Česen et al., 2013). In addition, it was shown that the initial phase of mitochondrial uncoupling is reversible for a considerable period,

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whereas LMP is irreversible and shortly followed by the plasma membrane rupture. However, LMP may be merely a bystander event and only a consequence of mitochondrial uncoupling (Giusti et al., 2009). These studies point to the complexity of cell death with regard to the organelle cross-talk and emphasize the need for parallel analysis of lysosomal, mitochondrial and plasma membrane integrity in order to comprehend it. Whereas endogenous apoptotic stimuli seem unlikely to primarily target lysosomes, targeting lysosomes with exogenous compounds is likely to have an early effect on lysosomes, although cell death may be mediated by unpredicted off-target (off-lysosome) effects. There are many forms of regulated necrosis and they participate in a variety of clinical disorders such as stroke, atherosclerosis, ischemia– reperfusion injury, pancreatitis, inflammatory bowel diseases, neurodegeneration and viral infections (Linkermann and Green, 2014; Vandenabeele et al., 2010). LMP is an early event in ischemia of cerebral tissue and cathepsin inhibitors considerably reduce cell damage, but it is poorly understood what mediates LMP and how LMP contributes to programmed neuronal necrosis (Lipton, 2013). It was postulated that increased Ca2 + concentration immediately after ischemia activates calpains which localize to the lysosomal membrane, where they cleave oxidation-modified Hsp70.1, which in turn destabilizes lysosomes and allows the release of cathepsins (Yamashima, 2012). The prototype of regulated necrosis is necroptosis, which is mediated through receptor-interacting protein kinase 1 (RIP1) (Vanden Berghe et al., 2010) or RIP3-MLKL (mixed lineage kinase domain-like) (Kaiser et al., 2013; Zhang et al., 2009) and which can be inhibited with the RIP1 inhibitor necrostatin-1 (Degterev et al., 2005). The execution of necroptosis involves a formation of a multiprotein complex containing RIP1 and RIP3, the so-called necrosome, which consequently leads to the active disintegration of mitochondria, lysosome and plasma membrane, and the activation of the immune system. In this sequence of events LMP occurs late in the process of the disintegration phase (Vanden Berghe et al., 2010; Vandenabeele et al., 2010). In addition, binding of RIP3 to necrosome triggers the activation of numerous enzymes involved in glycogen degradation, which augments glycolysis. Increased metabolism results in increased production of ROS that present increased risk for LMP. This is just one example how RIP3 and LMP are interconnected. In addition, ROS can also be generated on the plasma membrane, where NADPH oxidase 1 binds to TNFR and TNF-α stimulation results in NADPH oxidase activation and ROS production (Kreuzaler and Watson, 2012).

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which among others include caspase-8-mediated activation of acidsphingomyelinase (Werneburg et al., 2002) and c-jun N-terminal kinase-mediated PIXosome assembly on lysosomal membrane, which promotes insertion and oligomerization of BAX into the membrane (Guicciardi et al., 2013; Werneburg et al., 2012). Although LMP may occur upstream of MOMP in receptor-mediated apoptosis, the role of cysteine cathepsins does not seem critical, since gene ablation, knocking out, silencing or inhibition of cathepsins only has a minor effect on viability (Bojič et al., 2007; Klarič et al., 2009; Oberle et al., 2010; Spes et al., 2012; Vasiljeva et al., 2008; Wattiaux et al., 2007). However, LMP is readily observed at the late stage of apoptosis. It was shown that LMP occurs downstream of MOMP after Fas and etoposide treatment, UV radiation and interleukin-3 deprivation (Bojič et al., 2007; Oberle et al., 2010; Spes et al., 2012; Wattiaux et al., 2007). MOMP is accompanied by ROS generation (Turrens, 2003) and they are the most likely link to LMP, as discussed above.

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Contradicting observations and conclusions of in vitro cell experiments at least partially result from the different methods used, which seems to be a challenging subject. In spite of the relative abundance of lysosomes, evidence for LMP is therefore often not convincing and the nature of LMP is poorly defined. It is therefore important to overview the approaches used in the lysosome research in order to appropriately evaluate the functional consequences of LMP. Since there is a very dynamic and rapid cross-talk between different organelles and the associated processes potentially leading to cell death, it is of major importance to monitor in parallel the time course of LMP, MOMP, plasma membrane destabilization as well as of other cellular injuries. Pioneering studies of LMP were performed using isolated lysosomes (Goldman and Kaplan, 1973; Reeves, 1979). The system allows measurements of permeability for particular solutes in controllable conditions. However, long protocols for lysosome isolation can affect the permeability properties of isolated organelle membranes. In addition, luminal pH in isolated lysosomes may not be efficiently regulated, especially when lysosomes are exposed to amines, and this may affect the activity of intra-lysosomal enzymes as well as protonation and thereby the luminal retention of the compounds studied (Moriyama et al., 1992). Artificial membranes seem an attractive model system, but they lack the multitude of integral proteins present in real

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membranes and thereby the complexity, potentially leading to erroneous conclusions. Dialysis of the cytoplasm by transient permeabilization of the plasma membrane is technically demanding, but a relevant alternative because it preserves the complexity of the system (Steinberg et al., 2010). Most studies identifying LMP use cellular model systems and aim to establish the mode of action for the compound of interest. The first choice of assays is staining with lysosomotropic dyes, such as Lysotracker or acridine orange. However, the intrinsic problem with this assay when the dye fails to accumulate is that it does not discriminate between the alkanization of lysosomes due to the inactivity of the v-ATPase and the actual membrane permeabilization, which dissipates the proton gradient. So the next logical step is to look for the release of lysosomal luminal proteins into the cytosol, which has several constraints. The first concern is the selection of the marker. There are more than 50 different acid hydrolases and several activator proteins in the lumen of lysosomes (Schroder et al., 2010). Most of them are glycosylated, which substantially increases their molecular weight. Mature forms of cathepsins, which were shown to be able to initiate apoptotic signaling cascade if released into the cytosol (Cirman et al., 2004; Droga-Mazovec et al., 2008; Stoka et al., 2001), are 25–30 kDa molecules, which would correspond to a hydrodynamic radius of about 2.5 nm (Erickson, 2009). Many other hydrolases are even larger; acid ceramidase and acid phosphatase are about 50 kDa, whereas alpha-N-acetylglucosaminidase is about 80 kDa as a monomer, but also exists as a homodimer. The release of such big molecules would require large ‘gateways’ in the lysosomal membrane. In contrast, the loss of a proton gradient over the lysosomal/endosomal membrane can occur already after minor membrane destabilization. Second, the recovery mechanisms may erase traces of LMP. For example, unfolded enzymes may be degraded by the proteasome. Third, endocytic organelles together represent less than 10% of the cytoplasmic volume, so the content released into the cytosol becomes considerably diluted, which brings in the issue of the sensitivity of the methods. The two most common detection methods are enzyme activity measurement with chromogenic or fluorogenic synthetic substrates (Ivanova et al., 2008; Wilcox and Mason, 1992) and immunolabeling. Different concentrations of digitonin can be used to prepare cytosolic and total cell extracts, which can be probed for the presence of lysosomal enzymes either by activity measurements or immunolabeling (Appelqvist et al., 2011). Alternatively, cytosolic and total lysosomal enzyme activities can be measured directly in cells in which only plasma membrane or also intracellular membranes, respectively, are permeabilized with different digitonin concentrations (Foghsgaard et al., 2001; Groth-Pedersen et al., 2007; Hafner Česen et al., 2013). Immunocytochemistry allows subcellular localization of lysosomal markers in fixed and detergentpermeabilized cells. The loss of vesicular staining pattern and concomitant pale and diffuse staining of the cytosol monitored by antibody labeling would indicate the release of the lysosomal luminal content, but the quality of antibodies is a serious limiting factor (Helsby et al., 2013). Fluorescent activity-based probes bypass antibody restrictions and allow monitoring of enzyme localization in live or fixed cells (Blum et al., 2005; Chen et al., 2000; Edgington and Bogyo, 2013; Watzke et al., 2008). Another quite common approach is the release of fluorescent cargo molecules from pre-loaded lysosomes. Dextran molecules in the range of 10–70 kDa are used to determine the extent of membrane destabilization and the size of molecules that can be released into the cytosol. It is relevant to note that dextran molecules have larger hydrodynamic radii than proteins of the corresponding molecular weight (Armstrong et al., 2004; Erickson, 2009). Small dextran molecules and dyes, such as sulforhodamine with a molecular weight of about 600, are useful markers of moderate LMP (Steinberg et al., 2010). Even if the presence of lysosomal enzymes in the cytosol is convincingly demonstrated, it bears little information about the actual involvement of these enzymes in cell death signaling and cell demise. Selective small molecule inhibitors offer an attractive opportunity to address this

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of functional lysosomal cathepsins for downstream signaling (Hornung et al., 2008). Also cholesterol crystals can disrupt phagolysosmal membranes and activate the NLRP3 inflammasome. Moreover, the functional importance of LMP was demonstrated by the observations that cholesterol crystals injected intraperitoneally induce acute inflammation, which is impaired in mice deficient in components of the NLRP3 inflammasome, cathepsin B, cathepsin L or IL-1β molecules (Duewell et al., 2010). In addition to macrophages, NLRP3-mediated pyroptosis was observed also after LeuLeuOMe-induced lysosome destabilization in retinal pigment epithelial cells in a model of age-related macular degeneration. One possible agent that could destabilize lysosomes in this type of cells in vivo is the lipofuscin component A2E, which accumulates in lysosomes and has detergent-like properties (Tseng et al., 2013). There are also reports that argue for a minor role of LMP in pyroptosis signaling. It was recently reported that lysosome destabilization and the release of cysteine cathepsins is a late event in macrophages exposed to prototypical pyroptosis inducers and that NLRP3 inflammasome is activated before the lysosome rupture. In LeuLeuOMeor alum-treated cells lysosome destabilization triggered minimal capsase-1 activation and pyroptosis. Moreover, pro-forms of caspase1, IL-1β and IL-18 were degraded rather than proteolytically activated by cysteine cathepsins released into the cytosol, along with the degradation of other cytosolic proteins. This indicates that lysosome rupture might only be a weak activator of NLRP3 and caspase-1, which appears sufficient for promoting IL-1β release but not pyroptotic cell death (Lima et al., 2013). In macrophages treated with the NLRP3 activator nigericin, mitochondria were the first target; however, inflammasome activation was dependent on LMP, which was mediated by mitochondrial ROS (Heid et al., 2013). The observation that caspase-1-mediated increase in the cytosolic calcium concentration induces exocytosis of lysosomes in macrophages during pyroptosis, with the benefit of releasing antimicrobial factors into the extracellular space, is further supporting a nonessential role of LMP in NLRP3 activation (Bergsbaken et al., 2011). The above studies indicate that, as is the case with apoptosis, the lysosomal effectors that promote pyroptosis signaling are poorly understood. In order to sense LMP, NLRP3 might either act as a pattern recognition receptor that can directly bind various ligands, or detect an intermediate molecule that is generated by a common mechanism evoked by many different ligands (Hornung et al., 2008).

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For several years, the therapeutic potential of LMP to induce cell death of cancer cells has dominated the research in the field (Erdal et al., 2005; Kirkegaard and Jaattela, 2009) and a list of compounds, which induce LMP-dependent cell death, has grown (Boya, 2012). However, there is little understanding on the mechanisms connecting LMP with cell death signaling. Although Bid was found to be the major physiological substrate of lysosomal cathepsins, leading to mitochondrial cell death pathway, it is not the critical substrate (Cirman et al., 2004; Droga-Mazovec et al., 2008). Moreover, in several experimental models of LMP-mediated apoptosis or necrosis the downstream signaling is cathepsin-indepenedent (Melo et al., 2011; Rammer et al., 2010) and sensing of LMP by NLRP3 in LMPdependent pyroptosis is unknown (Hornung et al., 2008). In addition, it has been recognized that sub-lethal LMP can abolish the autophagic flux and so hamper recovery of the cell exposed to stress (Maclean et al., 2008; Repnik and Turk, 2010). Material scientists have joined the efforts to explore the potential of LMP for the release of drugs and genetic therapeutic agents into the cytosol via the endocytic pathway (Pack et al., 2005). This may revive the interest in the nature of LMP and its functional consequences with the prospect that multidisciplinary and interdisciplinary approaches would provide a solid data base for the design of novel therapeutic agents.

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We thank Gareth Griffiths for critical reading of the manuscript. The 674 work was supported by grants P1–0140, J1–4121 and J1–3602 from the 675 Slovene Research Agency to B.T. 676

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question. However, a serious problem of the inhibitors is their selectivity. Cysteine cathepsins are a good example. They have been shown to be able to cleave Bid to t-Bid and thus generate a signaling molecule for the intrinsic apoptotic pathway. However, synthetic cathepsin inhibitors above 1 μM concentration inhibit most of cysteine cathepsins, although enzyme kinetic parameters may indicate preferences for individual cysteine cathepsins in in vitro tests with recombinant cathepsins over a defined concentration range. It is therefore not appropriate to demonstrate cathepsin B involvement in cell death signaling on the basis of inhibition experiments with CA-074-OMe (Bogyo et al., 2000; Montaser et al., 2002). Furthermore, some cathepsin inhibitors such as E-64d also inhibit calpains, which makes interpretations difficult (Barrett et al., 1982). Therefore, if only one cysteine cathepsin inhibitor out of several tested is able to attenuate cell death, it raises doubt about the compound selectivity rather than provides evidence for the involvement of cysteine cathepsins. Even more inconvenient for cell death studies in cellular model systems is that several caspase inhibitors, including Z-DEVD-fmk and Z-VAD-fmk, considerably inhibit the activity of cysteine cathepsins at concentrations commonly used in cell death assays (Rozman-Pungerčar et al., 2003). Rather than with inhibitors, the role of individual cysteine cathepsins should be tested with siRNA or knock-out models, although the loss of one cathepsin may be compensated with the overexpression of others (Vasiljeva et al., 2008). In addition, cysteine cathepsins exhibit high redundancy in particular towards natural substrates, so it is not likely that one particular cleavage would be performed by a single cysteine cathepsin. This is well demonstrated by the ability of different cysteine cathepsins to process the Bid molecule at multiple cleavage sites (Droga-Mazovec et al., 2008; Repnik et al., 2012; Stoka et al., 2001). The ultimate challenge is to determine if LMP is the primary event in the cascade of cell death. It requires a parallel analysis of lysosomal, mitochondrial and plasma membrane integrity to make a comprehensive timeline of the events. If LMP is to be a starting point for the induction of cell death it is reasonable to expect that it happens early upon the uptake of the compound and well upstream of other injuries, including MOMP and plasma membrane rupture. This kinetic aspect of cell injuries is often overlooked and instead only a single time point at the late stage of cell death is analyzed, when cells are already severely damaged.

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