Lysosome-targeted stress reveals increased stability of lipofuscin-containing lysosomes
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Cellular ageing is associated with accumulation of undegradable intralysosomal material, called lipofuscin. In order to accelerate the lipofuscin accumulation, confluent, growth-arrested human fibroblasts were cultured under hyperoxic conditions. To provide a better insight into the effects of lipofuscin on cellular functions, we compared lysosomal stability in control and lipofuscin-loaded human fibroblasts under conditions of lysosome-targeted stress induced by exposure to either the lysosomotropic detergent MSDH or the redox-cycling quinone naphthazarin. We show that lysosomal damage, assessed by acridine-orange relocation, translocation of cathepsin D to the cytosol, and alkalinization of lysosomes, is more pronounced in control than in lipofuscin-loaded fibroblasts. Finding that lysosomal integrity was less affected or even preserved in case of lipofuscin-loaded cells enables us to suggest that lipofuscin exerts lysosome-stabilizing properties.
KeywordsAlkalinization Autophagolysosomes Bafilomycin A1 Cathepsin D MSDH Naphthazarin quinone
Membrane-bound acidic organelles, generally termed lysosomes, are considered a major site for degradation of both extra- and endogenously derived material (De Duve and Wattiaux 1966). In the latter case, degradation of the cell’s own constituents, so-called autophagy, not only serves nutritional purposes but also secures intracellular homeostasis through the removal of damaged and potentially hazardous biomolecules and organelles (Klionsky 2005). Along with the ability to provide for intracellular degradation and repair, the lysosomal system is also involved in different signal transduction pathways (Miaczynska et al. 2004), including that of programmed cell death (Ferri and Kroemer 2001; Yin et al. 2005). It has been shown that lysosome-targeted stress, induced by either the lysosomotropic detergent O-methyl-serine dodecylamide hydrochloride (MSDH; Li et al. 2000) or the redox-cycling quinone 5,8-dihydroxy-1,4-naphthoquinone (NzQ; Roberg et al. 1999), results in permeabilisation of lysosomal membrane, relocation of lysosomal constituents to the cytosol and, finally, apoptosis.
Another distinctive feature of lysosomal compartment is that it serves as a site for storage of non-degraded material. Such material is collectively called lipofuscin, when age-related, and ceroid when its accumulation is caused by pathological conditions (Porta 2002; Seehafer and Pearce 2006). According to the free-radical theory of ageing (Harman 1956), formation of oxidatively damaged intracellular structures is an inevitable side effect of aerobic life. Imperfect degradation of such damaged cellular components results in an accumulation of so-called “biological garbage” of which lipofuscin is an example (Brunk and Terman 2002a). A suitable model of induced cellular senescence is established by culturing cells under conditions of chronic oxidative stress, which accelerates age-related changes and results in premature lipofuscin accumulation (Grune et al. 2005; Terman and Brunk 1998). For the sake of simplicity, and due to the similarity of the mechanisms behind age-related and oxidative stress-induced accumulation (Brunk and Terman 2002a), we here refer to such intralysosomal pigment as lipofuscin.
Physiological effects of lipofuscin are generally viewed as deleterious due to the suggested implication of lipofuscin in the formation of free radicals (Brunk and Terman 2002b). It has been shown that lipofuscin acts as a photosensitizer, compromising the integrity of lysosomal membrane and finally resulting in cell death (Wihlmark et al. 1997). The age-related decline in the efficacy of certain proteases can also be explained in terms of lipofuscin effects. Thus, it has been suggested that newly produced lysosomal enzymes are misplaced to the lipofuscin-loaded lysosomes in a futile attempt to degrade lipofuscin instead of performing a useful function within autophagolysosomes (Terman and Brunk 2004). On the other hand, we recently found that moderate levels of lipofuscin are protective and increase the resistance of ageing fibroblasts to cell death (Stroikin et al. 2007). Beneficial effect of lipofuscin in this case is explained in terms of hormesis—an adaptation to low doses of otherwise harmful agents (Rattan 2004).
As has been recently shown by our group, complete starvation causes activation of programmed cell death through the destabilization of lysosomal compartment. Such a destabilization was suppressed in lipofuscin-loaded fibroblasts (Stroikin et al. 2007). The presented study is a continuation of the previous one in order to further investigate the possible hormetic effect of lipofuscin on lysosomal function. Instead of investigating the apoptotic response, which has been already found disturbed in lipofuscin-loaded cells, we focus on the stability of lysosomal compartment, considered one of the major regulators of programmed cell death. For this purpose, we compared the integrity of lysosomes in lipofuscin-loaded and control growth-arrested human fibroblasts under conditions of lysosome-targeted stress induced either by a lysosomotropic detergent (MSDH) or acute oxidative stress (NzQ). Here, we demonstrate that lysosome-targeted stress results in deleterious changes of lysosomal compartment that are significantly more pronounced in control fibroblasts than in lipofuscin-loaded cells.
Materials and methods
Culture conditions and experimental design
AG-1518 human fibroblasts (obtained from Coriell Institute, Camden, N.J., USA) were cultured in Eagle’s minimum essential medium supplemented with 10% foetal bovine serum, 2 mM glutamine, 100 IU/ml penicillin-G and 100 μg/ml streptomycin in an atmosphere of 8% O2, 87% N2 and 5% CO2, at 37°C (normal conditions). The cells were sub-cultivated at a 1:2 ratio until they reached passage 22–23, and were then allowed to grow until confluency. These cultures are referred to as control. Some confluent fibroblast cultures were exposed to 40% O2, 55% N2 and 5% CO2 (hyperoxia) for 2 months to induce lipofuscin accumulation (Terman and Brunk 1998) and are referred to as lipofuscin-loaded. The culture medium was changed twice a week. Evaluation of lipofuscin-accumulation was performed by flow cytometric estimation (Becton Dickinson Biosciences, San Jose, Calif., USA) of cellular autofluorescence and was found 2–3 fold higher in lipofuscin-loaded cells compared to controls (data not shown).
Both controls and lipofuscin-loaded cells were exposed to three different agents presumably affecting lysosomal integrity: (1) MSDH at a concentration of 25 μM for 15, 30 or 60 min; (2) NzQ at a concentration of 0.75 μM for 15, 30 or 60 min; (3) the vacuolar ATPase inhibitor bafilomycin A1 (Baf A1; Bowman et al. 1988) at a concentration of 20 nM for 15 or 30 min. Treatment with Baf A1 was used as a positive control during lysosomal pH assessment.
Lysosomal stability assessment by acridine orange (AO)
Lysosomal stability was assessed by the AO-relocation method (Olsson et al. 1989). AO is a lysosomotropic weak base with metachromatic features. Oligomeric form of highly concentrated and protonated AO (AOH+) exhibits red fluorescence, as is the case in intact lysosomes. Lysosomal alkalinization and translocation of lysosomal content to the cytosol during lysosomal stress results in the formation of the monomeric deprotonated form of AO with green fluorescence.
Cells on cover-slips were briefly stained with 5 μg/ml AO for 15 min under normal culture conditions, rinsed in complete medium and exposed to one of the lysosomal-stress-inducing agents as described in previous section. Live cultures were examined in an Axiovert S100TV microscope (Carl Zeiss, Jenna, Germany) equipped with a Hamamatsu digital camera C4742–95 (Hamamatsu, Hamamatsu City, Japan) using 60 x /1.4 oil lens, a halogen lamp and a blue-excitation filter. Emission was detected using a high pass filter above 520 nm. Phase-contrast and fluorescent images were obtained using OpenLab software (Improvision, Coventry, U.K.). Measurements of lysosomal red AO-fluorescence were performed using the National Institute of Health Image program (http://rsb.info.hih.gov/nih-image/). Fluorescence intensity was expressed in arbitrary units (a.u.) being a product of average pixel value per lysosome and the lysosome area.
Assessment of lysosomal pH
The lysosomal pH was measured by flow cytometry as described elsewhere (Nilsson et al. 2003). Briefly, cells were exposed to 40,000 MW FITC-dextran (Molecular Probes, Eugene, Ore., USA) at a concentration of 0.1 mg/ml for 3 days at 37°C. Loading with FITC-dextran was followed by incubation in complete medium for another 24 h. After that, cells were trypsinized, centrifuged at ∼300 g for 5 min, re-suspended in the culture medium, filtered through a 70-μm cell strainer and analyzed by flow cytometry (Becton Dickinson Biosciences). A 488-nm argon laser was used for the FITC excitation, and emission was detected in the FL1 and FL2 channels using a 530 ± 28-nm and a 610 ± 20-nm barrier filters, respectively. Data from 10,000 cells was analyzed using the CellQuest program (Becton Dickinson Immunocytometry systems). Modified Britton-Robinson buffers (pH 4.0–7.0), containing sodium azide and 2-deoxyglucose at a final concentration of 50 mM each and nigericin at a final concentration of 10 μM, were used for the preparation of a standard curve. The FL1/FL2 ratios were used to calculate the pH employing the standard curve.
Immunocytochemical detection of cathepsin D
Cells plated on cover-slips were fixed in 4% formaldehyde in phosphate-buffered saline (PBS) for 20 min in 4°C, rinsed in PBS, and exposed to incubation buffer containing 0.1% saponin and 5% foetal bovine serum in PBS for another 20 min at room temperature. The cells were then incubated with 1:100 diluted polyclonal rabbit anti-human antibodies to cathepsin D (DAKO, Roskilde, Denmark) for 1 h in a humidifier at room temperature. After rinsing in the incubation buffer (2 × 5 min), the specimens were exposed to 1:100 diluted goat anti-rabbit IgG-Alexa 594 conjugate (Molecular Probes) for 1 h at room temperature; rinsed in PBS and distilled water, and mounted in Vectashield (Vector Laboratories, Burlingame, Calif., USA). Images were obtained using a Microphot-SA fluorescence microscope equipped with an ORCA 100 Hamamatsu color digital camera (Hamamatsu).
Extraction of cytosol
Disturbed lysosomal integrity was additionally assessed by estimation of the amount of cathepsin D translocated to the cytosol during lysosomal stress. Cytosolic extraction was performed using the cholesterol-solubilizing agent digitonin as described elsewhere (Johansson et al. 2003). Briefly, digitonin at low concentrations permeabilizes cholesterol-rich membranes such as the plasma membrane, leaving cholesterol-poor membrane of lysosomes and mitochondria more or less intact. Cells were exposed to an extraction buffer (250 mM sucrose, 20 mM Hepes, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA and 1 mM EGTA) containing digitonin at concentration of 25 μg/ml on ice for 12 min. The extraction buffer was collected and proteins were precipitated by addition of 5% trichloric acid on ice for 10 min and centrifuged at ∼20,800 g for 15 min to obtain the protein pellet. Immunoblotting is described below.
Western blot analysis
The pellet was dissolved in 25 μl of lysis buffer containing 6 M urea, 63 mM Tris-HCl (pH 6.8), 10% glycerol and 2% SDS. Samples were then neutralized using 4 μl of 1 M NaOH per sample. Subsequently, 50 μM ditiotreitol (DTT) and 0.05% bromphenol blue were added and 30-μl aliquots of the cell lysate were fractionated by 15% SDS-PAGE. The proteins were then blotted on a nitrocellulose membrane, which was subsequently incubated in 5% skimmed milk in Tris-buffered saline (50 mM Tris, 0.15 M NaCl, pH 7.5) with 0.1% Tween-20 (TBS-T) for 90 min at room temperature and then washed in TBS-T. The membrane was then exposed overnight at 4°C to a monoclonal mouse anti-human cathepsin D antibody (Oncogene Research Products, San Diego, Calif., USA) diluted 1:1000 in 0.1% skimmed milk in TBS-T. The membrane was then washed and incubated for 1 h at room temperature with horseradish peroxidase (HRP)-conjugated goat anti-mouse secondary antibody (DAKO, Roskilde, Denmark) diluted 1:1500. Bands were visualized using Western blotting Luminol Reagent (Santa Cruz Biotechnology, Santa Cruz, Calif., USA). Equal loading was verified by reprobing the membrane with a mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Biogenesis, Poole, Dorset, U.K.) antibody diluted 1:400.
Transmission electron microscopy
Cells were fixed by adding 2% glutaraldehyde (Agar Scientic, Essex, U.K.) in 0.1 M sucrose-sodium cacodylate-HCl buffer (pH 7.2; Sigma, St Louis, Mo., USA) and post-fixed in osmium (Johnson Matthey Chemicals, Royston, U.K.). Specimens were stained en bloc with 2% uranyl acetate (Sigma) in 50% ethanol, dehydrated in a graded series of ethanol, embedded in Epon-812 (Fluka, Buchs, Switzerland) and polymerized at 60°C for 2 days. Thin sections were cut with a diamond knife (DIATOME, Bienne, Switzerland), stained with lead citrate, and then examined and photographed in a JEOL 1230-EX electron microscope (JEOL, Tokyo, Japan) at 100 kV.
For the estimation of AO-fluorescence, 100–140 lysosomes from each specimen were analyzed. All experiments were repeated at least three times. Values are given as means ±SD. Data obtained by the acridine orange relocation method were analyzed using the ANOVA and post hoc LSD test. The lysosomal pH data were analyzed using Kruskal–Wallis and post hoc Mann Whitney test. P values < 0.05 were considered significant.
Non-confocal fluorescent imaging of AO-stained cells shows that the number of lysosomes does not obviously differ between non-treated control and lipofuscin-loaded fibroblasts (Fig. 1b). Both MSDH- and NzQ-treatment result in decrease of the number of lysosomes in control cells, whereas neither of the treatments shows any effect on lysosomal number in lipofuscin-loaded fibroblasts (Fig. 1b).
First suggested by de Duve and Wattiaux (1966), the idea of lysosomal involvement in cell death has been gaining more evidence. During lysosome-targeted stress, the type of cell death depends on the degree of lysosomal damage. Thus, extensive release of lysosomal content results in necrosis, while partial lysosomal rupture mediates apoptosis (Brunk et al. 2001; Zhao et al. 2003). Lysosomal destabilization is an initial event in programmed cell death induced by oxidative stress, radiation, and exposure to MSDH and oxidized low-density lipoprotein (Brunk and Svensson 1999; Li et al. 2000; Persson et al. 2005; Yuan et al. 1997). It has been shown that permeabilisation of lysosomal membrane leading to translocation of lysosomal enzymes to the cytosol precedes mitochondrial release of cytochrome c and subsequent caspase-activation (Roberg et al. 1999).
The current study is a continuation of the recently presented work of our group, showing that lipofuscin-loaded cells are more resistant to cell death (Stroikin et al. 2007). Here, instead of studying the apoptotic response, we focus on the effects of lipofuscin on lysosomal stability, proven to be crucial for the initiation of apoptosis (Brunk et al. 2001).
The presented MSDH-induced vacuolization (Figs. 1a,b, and 2b) can be considered an adaptive cellular response as an attempt to limit the damage (Henics and Wheatley 1999) by means of autophagic sequestration. Since lysosomal enzymes translocated to the cytosol jeopardize survival of the cell, autophagy is primarily focused on sequestration of the released lysosomal content. It has also been suggested that damaged entire lysosomes can be autophagocytozed (Stroikin et al. 2004; Kiffin et al. 2006). Regardless of the mechanism of autophagic sequestration, it does not prevent apoptosis of MSDH-treated cells (Li et al. 2000), since the acidic interior of autophagolysosomes is attracting MSDH, which at low pH becomes protonated and acquires properties of a detergent (Firestone et al. 1979).
During MSDH-treatment, the number of vacuoles (Fig. 1a), corresponding to autophagolysosomes (Fig. 2b), becomes extensive, occupying the entire cell. This suggests that activated autophagy is no longer a repairing mechanism, but rather an executioner of cellular demise (Gozuacik and Kimchi 2004), which is in agreement with both the decrease of the amount of AO-positive lysosomes (Fig. 1b) and ultrastructural findings of the decreased number of morphologically typical lysosomes (Fig. 2a,b). These morphological features of lysosomal deterioration during MSDH-exposure are consistent with findings of gradual (1) decrease of lysosomal-associated AO-fluorescence, (2) increase of diffuse cathepsin D immunostaining, (3) increase of cathepsin D in the cytosolic fraction, and (4) lysosomal alkalinization. Although these changes are present in both control and lipofuscin-loaded fibroblasts, the deleterious effect of MSDH-treatment is significantly less pronounced in lipofuscin-loaded cells, which indicates reduced lysosomal sensitivity. The difference of susceptibility toward MSDH-induced lysosomal stress between control and lipofuscin-loaded fibroblasts cannot be explained in terms of decreased tropism of MSDH to lipofuscin-containing lysosomes, since lysosomal pH, responsible for both tropism and protonation of MSDH, is practically equally low in both control (4.24 ± 0.26) and lipofuscin-loaded (4.11 ± 0.17) cells. On the other hand, lipofuscin-containing lysosomes, when extensively overloaded, are excluded from physiological functioning as sites of degradation (Terman and Brunk 2004), causing formation of new, lipofuscin-free lysosomes. Such an increase of the overall volume of lysosomal compartment can be considered a possible modulator of reactivity towards lysosomotropic detergents. In this light, lipofuscin could be viewed as an indirect factor of increasing lysosomal stability. But since the number of lysosomes in non-treated cells does not markedly differ between control and lipofuscin-loaded fibroblasts (Fig. 1b), the volume of the lysosomal compartment cannot be rendered as a factor responsible for the increased resistance to MSDH-treatment in the presented study.
During NzQ-treatment of control cells, the agglomeration of lysosomes without apparent cytosolic vacuolization (Fig. 1a) might represent successful reparative autophagy. Lysosomal damage revealed by the initial decrease of lysosomal fluorescence (Fig. 3b), translocation of cathepsin D to the cytosol (Figs. 4 and 5) and lysosomal alkalinization (Fig. 6b) triggers the mechanisms leading to the accomplished control over the damage. The idea of effective autophagic repair is consistent with the results showing the eventual (1) regaining of lysosomal-associated AO-fluorescence (Fig. 3b), (2) recovery of the grainy pattern of cathepsin D immunostaining (Fig. 4) (3) decrease of the cytosolic fraction of cathepsin D (Fig. 5), and (4) lysosomal acidification (Fig. 6b) of control fibroblasts after 60 min of exposure to NzQ. Logically, not all cells manage to accomplish a successful damage-control. Some cells die due to the extensive lysosomal damage. Considering that one fraction of cells die, measurements performed on NzQ-exposed cells might represent cellular resistance rather than recovery from cell damage. On the other hand, during continuous exposure to NzQ, lysosomal agglomeration (Fig. 1a), coexisting with a decrease of the number of lysosomes (Fig. 1b), suggests an ongoing intracellular process by which cells cope with unfavorable conditions. Even if some cells are lost during NzQ-treatment, conclusions drawn from the measurements made on the surviving population of cells still emphasize the mechanism of cell survival.
The absence of changes in lysosomal compartment of lipofuscin-loaded cells suggests that NzQ-treatment does not cause significant lysosomal damage in these cells. According to recent theories, high content of iron associated with lipofuscin should have increased the lysosomal sensitivity to oxidative stress (Yu et al. 2003). Alternatively, the resistance of lipofuscin-loaded cells can be due to their pre-exposure to hyperoxic conditions. The possibility of up-regulation of anti-oxidative defense during chronic oxidative stress still remains to be investigated.
Physiological effects of lipofuscin on cellular functions in general, and on lysosomal integrity in particular, have been a matter of controversial opinions. The increased sensitivity of lipofuscin-accumulating cells to lysosomal breach and apoptosis has been explained in terms of redox-active iron content, which promotes formation of free radicals under conditions of oxidative stress (Terman and Brunk 2004). In opposition, some researchers doubt that a convincing evidence of deleterious effects of lipofuscin has ever been demonstrated (Porta et al. 2002). Moreover, we recently showed that moderate levels of lipofuscin have protective effects on cell survival during nutritional deprivation (Stroikin et al. 2007). The previously suggested idea, that lipofuscin permanently occupies active sites of lysosomal enzymes, preventing their translocation to the cytosol and engagement in programmed cell death during lysosomal stress, is consistent with the present findings of decreased cytosolic translocation of cathepsin D in lipofuscin-loaded cells. In addition, lipofuscin exhibits some proton-trapping properties, considering that decrease of the proton gradient upon treatment with Baf A1 is significantly lower in lipofuscin-loaded cells (Fig. 6c). Positive correlation between cellular lipofuscin content and resistance to oxidative stress can also be indicative of lipofuscin functioning as a trap for free radicals, explaining the high resistance of lipofuscin-loaded cells to NzQ-treatment. While the exact mechanism of lipofuscin influence on cellular functions remains to be elucidated, we suggest that lipofuscin has lysosome-stabilizing properties, making these organelles less sensitive and diminishing their influence on cellular functioning.
In conclusion, increased autophagy following MSDH-treatment can be viewed as an adaptive cellular response in order to limit the damage caused by the leakage of protons and proteolytic enzymes into the cytosol because of impairment of the lysosomal membrane. Relocation of lysosomal content to the cytosol and decrease of intralysosomal pH are indicators of lysosome-targeted stress induced either by exposure to the lysosomotropic detergent MSDH or the redox-cycling quinone naphthazarin. Such a destabilization is significantly hampered in lysosomes, which contain the ageing-associated pigment lipofuscin. Increased stability of lipofuscin-containing lysosomes can be related to the decreased inducibility of apoptosis in ageing cells.
We thank Linda Vainikka for technical assistance. This study was financially supported by Lions Research Foundation and by grant from the Medical Branch of the Swedish Research Council (Vetenskapsrådet).
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