Karin Roberg

Regulation of apoptosis-associated lysosomal membrane permeabilization

Lysosomal membrane permeabilization (LMP) occurs in response to a large variety of cell death stimuli causing release of cathepsins from the lysosomal lumen into the cytosol where they participate in apoptosis signaling. In some settings, apoptosis induction is dependent on an early release of cathepsins, while under other circumstances LMP occurs late in the cell death process and contributes to amplification of the death signal. The mechanism underlying LMP is still incompletely understood; however, a growing body of evidence suggests that LMP may be governed by several distinct mechanisms that are likely engaged in a death stimulus- and cell-type-dependent fashion. In this review, factors contributing to permeabilization of the lysosomal membrane including reactive oxygen species, lysosomal membrane lipid composition, proteases, p53, and Bcl-2 family proteins, are described. Potential mechanisms to safeguard lysosomal integrity and confer resistance to lysosome-dependent cell death are also discussed.

Photo-oxidative disruption of lysosomal membranes causes apoptosis of cultured human fibroblasts.

Acridine orange (AO) is a lysosomotropic weak base, a metachromatic fluorochrome, and a photosensitizer, as well. Living cells that are exposed for a short period of time to this compound at low concentration, and under ordinary culture conditions, accumulate the drug within their acidic vacuolar compartment, giving rise to a mainly red, granular fluoresence upon excitation with blue light. When AO-loaded cells are irradiated with intense blue light, AO soon starts to leak from late endosomes and lysosomes, partially shifting the fluorescence to a green, nuclear and diffuse cytosolic, one. This AO-relocalization is a consequence of photo-oxidation of the lysosomal membranes, which initially results in disruption of their proton-gradients and later, in leakage into the cytosol of a host of hydrolytic enzymes--as was here demonstrated by immunocytochemistry--which are capable of causing cellular damage. Most fibroblasts survived minor photo-oxidation, with a period of reparative autophagocytosis. Severe photo-oxidation, which resulted in severe lysosomal damage, caused cellular necrosis; whereas moderate stress, resulting in only partial lysosomal leakiness lead to apoptosis with TUNEL-positive nuclei and shrunken cytoplasm. The findings of the present study show that photo-oxidative damage to the membranes that surround the acidic vacuolar compartment, is an event that results in release of proteolytic and DNA-fragmenting enzymes into the cytosol, which may induce either necrosis, apoptosis, or reparable sublethal damage, depending on the magnitude of lysosomal rupture. Furthermore, the results strongly suggest that proteases and endonucleases of lysosomal origin may induce apoptosis if relocalized from the acidic vacuolar compartment into the cytosol.

Lysosomal release of Cathepsin D precedes relocation of Cytochrome C and loss of mitochondrial transmembrane potential during apoptosis induced by oxidative stress

Apoptosis was induced in human foreskin fibroblasts by the redox-cycling quinone naphthazarin (5,8-dihydroxy-1,4-naphthoquinone). Most of the cells displayed ultrastructure typical of apoptosis after 8 h of exposure to naphthazarin. Apoptosis was inhibited in fibroblasts pretreated with the cathepsin D inhibitor pepstatin A. Immunofluorescence analysis of the intracellular distribution of cathepsin D revealed a distinct granular pattern in control cells, whereas cells treated with naphthazarin for 30 min exhibited more diffuse staining that corresponded to release of the enzyme from lysosomes to the cytosol. After 2 h, release of cytochrome c from mitochondria to the cytosol was indicated by immunofluorescence. The membrane-potential-sensitive probe JC-1 and flow cytometry did not detect a permanent decrease in mitochondrial transmembrane potential (delta psi(m)) until after 5 h of naphthazarin treatment. Our findings show that, during naphthazarin-induced apoptosis, lysosomal destabilization (measured as release of cathepsin D) precedes release of cytochrome c, loss of delta psi(m), and morphologic alterations. Moreover, apoptosis could be inhibited by pretreatment with pepstatin A.

Cathepsin D mediates cytochrome c release and caspase activation in human fibroblast apoptosis induced by staurosporine

AbstractThere is increasing evidence that proteases other than caspases, for example, the lysosomal cathepsins B, D and L, are involved in apoptotic cell death. In the present study, we present data that suggest a role for cathepsin D in staurosporine-induced apoptosis in human foreskin fibroblasts. Cathepsin D and cytochrome c were detected partially released to the cytosol after exposure to 0.1 μM staurosporine for 1 h. After 4 h, activation of caspase-9 and -3 was initiated and later caspase-8 activation and a decrease in full-length Bid were detected. Pretreatment of cells with the cathepsin D inhibitor, pepstatin A, prevented cytochrome c release and caspase activation, and delayed cell death. These results imply that cytosolic cathepsin D is a key mediator in staurosporine-induced apoptosis. Analysis of the relative sequence of apoptotic events indicates that, in this cell type, cathepsin D acts upstream of cytochrome c release and caspase activation.

Microinjection of cathepsin D induces caspase-dependent apoptosis in fibroblasts

Recent reports have indicated that enzymes such as cathepsins D and B are translocated from lysosomal compartments to the cytosol early during apoptosis. We have previously noted that a translocation of cathepsins D and B occur before cytochrome c release and caspase activation in cardiomyocytes and human fibroblasts during oxidative stress-induced apoptosis. In the present report, we use a microinjection technique to investigate if cytosolic location of the cathepsins D and B are important for induction of apoptosis. We found that microinjection of cathepsin D into the cytosol of human fibroblasts caused apoptosis, which was detected as changes in distribution of cytochrome c, cell shrinkage, activation of caspases, chromatin condensation, and formation of pycnotic nuclei. No apoptosis was, however, induced by microinjection of cathepsin B. Moreover, apoptosis was prevented in fibroblasts pretreated with a caspase-3-like inhibitor, and also when microinjected with cathepsin D mixed with the cathepsin D inhibitor, pepstatin A. These results show that cytosolic cathepsin D can act as a proapoptotic mediator upstream of cytochrome c release and caspase activation in human fibroblasts.

Interconnections between apoptotic, autophagic and necrotic pathways: implications for cancer therapy development

The rapid accumulation of knowledge on apoptosis regulation in the 1990s was followed by the development of several experimental anticancer‐ and anti‐ischaemia (stroke or myocardial infarction) drugs. Activation of apoptotic pathways or the removal of cellular apoptotic inhibitors has been suggested to aid cancer therapy and the inhibition of apoptosis was thought to limit ischaemia‐induced damage. However, initial clinical studies on apoptosis‐modulating drugs led to unexpected results in different clinical conditions and this may have been due to co‐effects on non‐apoptotic interconnected cell death mechanisms and the ‘yin‐yang’ role of autophagy in survival versus cell death. In this review, we extend the analysis of cell death beyond apoptosis. Upon introduction of molecular pathways governing autophagy and necrosis (also called necroptosis or programmed necrosis), we focus on the interconnected character of cell death signals and on the shared cell death processes involving mitochondria (e.g. mitophagy and mitoptosis) and molecular signals playing prominent roles in multiple pathways (e.g. Bcl2‐family members and p53). We also briefly highlight stress‐induced cell senescence that plays a role not only in organismal ageing but also offers the development of novel anticancer strategies. Finally, we briefly illustrate the interconnected character of cell death forms in clinical settings while discussing irradiation‐induced mitotic catastrophe. The signalling pathways are discussed in their relation to cancer biology and treatment approaches.

H2O2-Mediated Damage to Lysosomal Membranes of J-774 Cells

The effects of hydrogen peroxide on cell viability and, in particular, on lysosomal integrity were investigated in a model system of cultured, established, macrophage-like J-774 cells. The cells were found to rapidly degrade added hydrogen peroxide, withstanding concentrations < or = 250 microM without cell death; however, all tested concentrations (100-500 microM) substantially decreased cellular ATP to approximately the same degree. Concentrations of hydrogen peroxide > or = 500 microM resulted in a pronounced and rapid decrease in cell viability preceded by the loss of lysosomal integrity, as judged by the relocalization of acridine orange, a lysosomotropic weak base, in pre-labelled cells. Hydrogen peroxide-induced relocalization of acridine orange and cell death were either enhanced or much prevented, according to if the cells were initially allowed to endocytose ferric iron or the specific iron-chelator deferoxamine, respectively. Depletion of ATP, however, was not associated with the loss of lysosomal integrity and viability regardless of iron or deferoxamine pretreatment. Pre-exposure to E-64, an inhibitor of lysosomal thiol proteases, resulted in the reduction of both lysosomal membrane damage and cell death. The results are interpreted as indicating (i) generation of hydroxyl radicals within the secondary lysosomal compartment due to the occurrence of reactive ferrous iron, leading to (ii) peroxidative alterations of the lysosomal membrane resulting in (iii) loss of lysosomal membrane integrity with dissipation of the proton gradient and leakage of lysosomal contents, including hydrolytic enzymes, into the cell sap.

Lipofuscin accumulation in cultured retinal pigment epithelial cells causes enhanced sensitivity to blue light irradiation.

Lipofuscin accumulates with age within secondary lysosomes of retinal pigment epithelial (RPE) cells of humans and many animals. The autofluorescent lipofuscin pigment has an excitation maximum within the range of visible blue light, while it is emitting in the yellow-orange area. This physico-chemical property of the pigment indicates that it may have a photo-oxidative capacity and, consequently, then should destabilize lysosomal membranes of blue-light exposed RPE. To test this hypothesis, being of relevance to the understanding of age-related macular degeneration, cultures of heavily lipofuscin-loaded RPE cells were blue-light-irradiated and compared with respect to lysosomal stability and cell viability to relevant controls. To rapidly convert primary cultures of RPE, obtained from neonatal rabbits, into aged, lipofuscin-loaded cells, they were allowed to phagocytize artificial lipofuscin that was prepared from outer segments of bovine rods and cones. Following blue-light irradiation, lysosomal membrane stability was measured by vital staining with the lysosomotropic weak base, and metachromatic fluorochrome, acridine orange (AO). Quantifying red (high AO concentration within intact lysosomes with preserved proton gradient over their membranes) and green fluorescence (low AO concentration in nuclei, damaged lysosomes with decreased or lost proton gradients, and in the cytosol) allowed an estimation of the lysosomal membrane stability after blue-light irradiation. Cellular viability was estimated with the delayed trypan blue dye exclusion test. Lipofuscin-loaded blue-light-exposed RPE cells showed a considerably enhanced loss of both lysosomal stability and viability when compared to control cells. It is concluded that the accumulation of lipofuscin within secondary lysosomes of RPE sensitizes these cells to blue light by inducing photo-oxidative alterations of their lysosomal membranes resulting in a presumed leakage of lysosomal contents to the cytosol with ensuing cellular degeneration of apoptotic type. The suggested mechanism may have bearings on the development of age-related macular degeneration.

Lysosomal membrane permeabilization during apoptosis--involvement of Bax?

Bcl‐2 family members have long been known to control permeabilization of the mitochondrial membrane during apoptosis, but involvement of these proteins in lysosomal membrane permeabilization (LMP) was not considered until recently. The aim of this study was to investigate the mechanism underlying the release of lysosomal proteases to the cytosol seen during apoptosis, with special emphasis on the role of Bax. In human fibroblasts, exposed to the apoptosis‐inducing drug staurosporine (STS), the release of the lysosomal protease cathepsin D to the cytosol was observed by immunocytochemistry. In response to STS treatment, there was a shift in Bax immunostaining from a diffuse to a punctate pattern. Confocal microscopy showed co‐localization of Bax with both lysosomes and mitochondria in dying cells. Presence of Bax at the lysosomal membrane was confirmed by immuno‐electron microscopy. Furthermore, when recombinant Bax was incubated with pure lysosomal fractions, Bax inserted into the lysosomal membrane and induced the release of lysosomal enzymes. Thus, we suggest that Bax is a mediator of LMP, possibly promoting the release of lysosomal enzymes to the cytosol during apoptosis.

Relocalization of Cathepsin D and Cytochrome c Early in Apoptosis Revealed by Immunoelectron Microscopy

Cathepsin D was translocated from lysosomal structures to the cytosol in primary cultures of neonatal rat cardiomyocytes exposed to oxidative stress, and these cells underwent apoptotic death during subsequent incubation. Temporal aspects of cathepsin D relocalization, cytochrome c release, and decrease in mitochondrial transmembrane potential (Δψm) were studied in myocytes exposed to the redox-cycling xenobiotic naphthazarin (5,8-dihydroxy-1,4-naphthoquinone). Immunofluorescence labeling revealed that cathepsin D was translocated to the cytosol after 30 minutes of naphthazarin treatment, and cytochrome c was released from mitochondria to the cytosol after 2 hours. Western blotting and immunoelectron microscopy indicated a minor release of cytochrome c after only 30 minutes and 1 hour, respectively. Thereafter, a decrease in Δψm was detected using the Δψm-sensitive dye JC-1 and confocal microscopy, and ultrastructural analysis indicated apoptotic morphology. Pretreatment of the cultures with the cathepsin D inhibitor pepstatin A prevented release of cytochrome c from mitochondria and maintained the Δψm. Moreover, ultrastructural examination showed no apoptotic morphology. These findings suggest that lysosomal destabilization (detected as the release of cathepsin D) and release of cytochrome c from mitochondria take place early in apoptosis. Also, the former event probably occurs before the latter during apoptosis induced by oxidative stress because pretreatment with pepstatin A prevented release of cytochrome c and loss of Δψm in cardiomyocytes exposed to naphthazarin.