Cell sensitivity to oxidative stress is influenced by ferritin autophagy

To test the consequences of lysosomal degradation of differently iron-loaded ferritin molecules and to mimic ferritin autophagy under iron-overload and normal conditions, J774 cells were allowed to endocytose heavily iron loaded ferritin, probably with some adventitious iron (Fe-Ft), or iron-free apo-ferritin (apo-Ft). When cells subsequently were exposed to a bolus dose of hydrogen peroxide, apo-Ft prevented lysosomal membrane permeabilization (LMP), whereas Fe-Ft enhanced LMP. A 4-h pulse of Fe-Ft initially increased oxidative stress-mediated LMP that was reversed after another 3h under standard culture conditions, suggesting that lysosomal iron is rapidly exported from lysosomes, with resulting upregulation of apo-ferritin that supposedly is autophagocytosed, thereby preventing LMP by binding intralysosomal redox-active iron. The obtained data suggest that upregulation of the stress protein ferritin is a rapid adaptive mechanism that counteracts LMP and ensuing apoptosis during oxidative stress. In addition, prolonged iron starvation was found to induce apoptotic cell death that, interestingly, was preceded by LMP, suggesting that LMP is a more general phenomenon in apoptosis than so far recognized. The findings provide new insights into aging and neurodegenerative diseases that are associated with enhanced amounts of cellular iron and show that lysosomal iron loading sensitizes to oxidative stress.     

Sphingosine mediates TNFα-induced lysosomal membrane permeabilization and ensuing programmed cell death in hepatoma cells

Normally, cell proliferation and death are carefully balanced in higher eukaryotes, but one of the most important regulatory mechanisms, apoptosis, is upset in many malignancies, including hepatocellular-derived ones. Therefore, reinforcing cell death often is mandatory in anticancer therapy. We previously reported that a combination of tumor necrosis factor-α (TNF) and cycloheximide (CHX) efficiently kill HTC cells, a rat hepatoma line, in an apoptosis-like mode. Death is actively mediated by the lysosomal compartment, although lysosomal ceramide was previously shown not to be directly implicated in this process. In the present study, we show that TNF/CHX increase lysosomal ceramide that is subsequently converted into sphingosine. Although ceramide accumulation does not significantly alter the acidic compartment, the sphingosine therein generated causes lysosomal membrane permeabilization (LMP) followed by relocation of lysosomal cathepsins to the cytoplasm. TNF/CHX-induced LMP is effectively abrogated by siRNAs targeting acid sphingomyelinase or acid ceramidase, which prevent both LMP and death induced by TNF/CHX. Taken together, our results demonstrate that lysosomal accumulation of ceramide is not detrimental per se, whereas its degradation product sphingosine, which has the capacity to induce LMP, appears responsible for the observed apoptotic-like death.     

The lysosomal-mitochondrial axis theory of postmitotic aging and cell death

Aging (senescence) is characterized by a progressive accumulation of macromolecular damage, supposedly due to a continuous minor oxidative stress associated with mitochondrial respiration. Aging mainly affects long-lived postmitotic cells, such as neurons and cardiac myocytes, which neither divide and dilute damaged structures, nor are replaced by newly differentiated cells. Because of inherent imperfect lysosomal degradation (autophagy) and other self-repair mechanisms, damaged structures (biological "garbage") progressively accumulate within such cells, both extra- and intralysosomally. Defective mitochondria and aggregated proteins are the most typical forms of extralysosomal "garbage", while lipofuscin that forms due to iron-catalyzed oxidation of autophagocytosed or heterophagocytosed material, represents intralysosomal "garbage". Based on findings that autophagy is diminished in lipofuscin-loaded cells and that cellular lipofuscin content positively correlates with oxidative stress and mitochondrial damage, we have proposed the mitochondrial-lysosomal axis theory of aging, according to which mitochondrial turnover progressively declines with age, resulting in decreased ATP production and increased oxidative damage. Due to autophagy of ferruginous material, lysosomes contain a pool of redox-active iron, which makes these organelles particularly susceptible to oxidative damage. Oxidant-mediated destabilization of lysosomal membranes releases hydrolytic enzymes to the cytosol, eventuating in cell death (either apoptotic or necrotic depending on the magnitude of the insult), while chelation of the intralysosomal pool of redox-active iron prevents these effects. In relation to the onset of oxidant-induced apoptosis, but after the initiating lysosomal rupture, cytochrome c is released from mitochondria and caspases are activated. Mitochondrial damage follows the release of lysosomal hydrolases, which may act either directly or indirectly, through activation of phospholipases or pro-apoptotic proteins such as Bid. Additional lysosomal rupture seems to be a consequence of a transient oxidative stress of mitochondrial origin that follows the attack by lysosomal hydrolases and/or phospholipases, creating an amplifying loop system.     

Intracellular free iron and acidic pathways mediate TNF-induced death of rat hepatoma cells

Rat hepatoma HTC cells are intrinsically resistant to various apoptosis-inducing agents. Strategies to induce death in hepatoma cells are needed and the present experimental study was aimed to investigate the sensitivity of HTC cells to TNF and to clarify the mechanisms of action of this cytokine. Cells were treated with TNF and death mechanisms characterized employing an integration of morphological and biochemical techniques. HTC cells, sensitized to TNF toxicity with cycloheximide, died in a caspase-independent apoptosis-like manner. Although we found no evidence for a direct involvement of lysosomal cathepsins, bafilomycin A1 and ammonium chloride significantly attenuated TNF toxicity. Also desferrioxamine mesylate, an iron chelator, partly protected the cells from TNF, while a complete protection was afforded by combining ammonium chloride and iron chelator. Moreover, HTC were protected from TNF also by lipophylic antioxidants and diphenylene iodonium chloride, a NADPH oxidase inhibitor. These data depict a novel mechanism of TNF-mediated cytotoxicity in HTC cells, in which the endo-lysosomal compartment, NADPH oxidase and an iron-mediated pro-oxidant status contribute in determining a caspase-independent, apoptosis-like cell death.     

Radiation-induced lysosomal iron reactivity: implications for radioprotective therapy

A novel mechanism of radiosensitization involves radiation-enhanced autophagy of damaged mitochondria and various metalloproteins, by which iron accumulates within lysosomes. Hydrogen peroxide, formed by the radiolytic cleavage of water, generates in the presence of lysosomal redox-active iron extremely reactive hydroxyl radicals by Fenton-type chemistry. Subsequent peroxidative damage of lysosomal membranes initiates release of harmful content from ruptured lysosomes that triggers a cascade of events eventuating in DNA damage and apoptotic or necrotic cell death. This article reviews the role of lysosomal destabilization in radiation-induced cell damage and death. The potential effects of iron chelation therapy targeted to the lysosomes for protection of normal tissues against unwanted effects by radiation is also discussed.     

-Lipoic acid and -lipoamide prevent oxidant-induced lysosomal rupture and apoptosis

Abstract

-Lipoic acid (LA) and its corresponding derivative, -lipoamide (LM), have been described as antioxidants, but the mechanisms of their putative antioxidant effects remain largely uncharacterised. The vicinal thiols present in the reduced forms of these compounds suggest that they might possess metal chelating properties. We have shown previously that cell death caused by oxidants may be initiated by lysosomal rupture and that this latter event may involve intralysosomal iron which catalyzes Fenton-type chemistry and resultant peroxidative damage to lysosomal membranes. Here, using cultured J774 cells as a model, we show that both LA and LM stabilize lysosomes against oxidative stress, probably by chelating intralysosomal iron and, consequently, preventing intralysosomal Fenton reactions. In preventing oxidant-mediated apoptosis, LM is significantly more effective than LA, as would be expected from their differing capacities to enter cells and concentrate within the acidic lysosomal compartment. As previously reported, the powerful iron-chelator, desferrioxamine (Des) (which also locates within the lysosomal compartment), also provides protection against oxidant-mediated cell death. Interestingly, although Des enhances the partial protection afforded by LA, it confers no additional protection when added with LM. Therefore, the antioxidant actions of LA and LM may arise from intralysosomal iron chelation, with LM being more effective in this regard.     

Lysosomal heterogeneity between and within cells with respect to resistance against oxidative stress

The prevailing opinion on lysosomal endurance is that, as long as the cells are still alive, these organelles are generally quite stable and, thus, do not induce cell damage by leaking their numerous powerful hydrolytic enzymes to the cytosol. We suggest that this opinion is basically wrong and consider that many lysosomes are quite vulnerable, especially to oxidative stress. Moreover, we suggest that cellular degeneration, including apoptosis as well as necrosis, follows upon lysosomal disruption. We have found differing stability of lysosomal membranes to oxidative stress, not only among different cell types, but also between cells of the same type and between lysosomes of individual cells. We suggest that cellular resistance to oxidative stress is mainly a function of three parameters: (i) the capacity to degrade hydrogen peroxide before it reaches, and may diffuse into, the acidic vacuolar compartment; (ii) the resistance to reactive oxygen species of lysosomal membranes; and (iii) the intralysosomal amounts of redox-active, low molecular weight iron. Iron-catalysed intralysosomal reactions, if pronounced enough, result in peroxidation and destabilization of the lysosomal membrane. Owing to differences in the cellular synthesis of hydrogen peroxide-degrading enzymes, degree of autophagocytotic degradation of iron-containing metalloproteins, lysosomal localization within the cytoplasm and intralysosomal iron chelation, the above three parameters may vary between both different and similar cells and between lysosomes of individual cells as well, explaining their observed variability with respect to resistance against oxidative stress     

Lysosomal heterogeneity between and within cells with respect to resistance against oxidative stress

The prevailing opinion on lysosomal endurance is that, as long as the cells are still alive, these organelles are generally quite stable and, thus, do not induce cell damage by leaking their numerous powerful hydrolytic enzymes to the cytosol. We suggest that this opinion is basically wrong and consider that many lysosomes are quite vulnerable, especially to oxidative stress. Moreover, we suggest that cellular degeneration, including apoptosis as well as necrosis, follows upon lysosomal disruption. We have found differing stability of lysosomal membranes to oxidative stress, not only among different cell types, but also between cells of the same type and between lysosomes of individual cells. We suggest that cellular resistance to oxidative stress is mainly a function of three parameters: (i) the capacity to degrade hydrogen peroxide before it reaches, and may diffuse into, the acidic vacuolar compartment; (ii) the resistance to reactive oxygen species of lysosomal membranes; and (iii) the intralysosomal amounts of redox-active, low molecular weight iron. Iron-catalysed intralysosomal reactions, if pronounced enough, result in peroxidation and destabilization of the lysosomal membrane. Owing to differences in the cellular synthesis of hydrogen peroxide-degrading enzymes, degree of autophagocytotic degradation of iron-containing metalloproteins, lysosomal localization within the cytoplasm and intralysosomal iron chelation, the above three parameters may vary between both different and similar cells and between lysosomes of individual cells as well, explaining their observed variability with respect to resistance against oxidative stress This revised version was published online in November 2006 with corrections to the Cover Date.     

Lysosomal heterogeneity between and within cells with respect to resistance against oxidative stress

The prevailing opinion on lysosomal endurance is that, as long as the cells are still alive, these organelles are generally quite stable and, thus, do not induce cell damage by leaking their numerous powerful hydrolytic enzymes to the cytosol. We suggest that this opinion is basically wrong and consider that many lysosomes are quite vulnerable, especially to oxidative stress. Moreover, we suggest that cellular degeneration, including apoptosis as well as necrosis, follows upon lysosomal disruption. We have found differing stability of lysosomal membranes to oxidative stress, not only among different cell types, but also between cells of the same type and between lysosomes of individual cells. We suggest that cellular resistance to oxidative stress is mainly a function of three parameters: (i) the capacity to degrade hydrogen peroxide before it reaches, and may diffuse into, the acidic vacuolar compartment; (ii) the resistance to reactive oxygen species of lysosomal membranes; and (iii) the intralysosomal amounts of redox-active, low molecular weight iron. Iron-catalysed intralysosomal reactions, if pronounced enough, result in peroxidation and destabilization of the lysosomal membrane. Owing to differences in the cellular synthesis of hydrogen peroxide-degrading enzymes, degree of autophagocytotic degradation of iron-containing metalloproteins, lysosomal localization within the cytoplasm and intralysosomal iron chelation, the above three parameters may vary between both different and similar cells and between lysosomes of individual cells as well, explaining their observed variability with respect to resistance against oxidative stress This revised version was published online in November 2006 with corrections to the Cover Date.     

Intralysosomal iron: a major determinant of oxidant-induced cell death

As a result of continuous digestion of iron-containing metalloproteins, the lysosomes within normal cells contain a pool of labile, redox-active, low-molecular-weight iron, which may make these organelles particularly susceptible to oxidative damage. Oxidant-mediated destabilization of lysosomal membranes with release of hydrolytic enzymes into the cell cytoplasm can lead to a cascade of events eventuating in cell death (either apoptotic or necrotic depending on the magnitude of the insult). To assess the importance of the intralysosomal pool of redox-active iron, we have temporarily blocked lysosomal digestion by exposing cells to the lysosomotropic alkalinizing agent, ammonium chloride (NH(4)Cl). The consequent increase in lysosomal pH (from ca. 4.5 to > 6) inhibits intralysosomal proteolysis and, hence, the continuous flow of reactive iron into this pool. Preincubation of J774 cells with 10 mM NH(4)Cl for 4 h dramatically decreased apoptotic death caused by subsequent exposure to H(2)O(2), and the protection was as great as that afforded by the powerful iron chelator, desferrioxamine (which probably localizes predominantly in the lysosomal compartment). Sulfide-silver cytochemical detection of iron revealed a pronounced decrease in lysosomal content of redox-active iron after NH(4)Cl exposure, probably due to diminished intralysosomal digestion of iron-containing material coupled with continuing iron export from this organelle. Electron paramagnetic resonance experiments revealed that hydroxyl radical formation, readily detectable in control cells following H(2)O(2) addition, was absent in cells preexposed to 10 mM NH(4)Cl. Thus, the major pool of redox-active, low-molecular-weight iron may be located within the lysosomes. In a number of clinical situations, pharmacologic strategies that minimize the amount or reactivity of intralysosomal iron should be effective in preventing oxidant-induced cell death.     

Sensitive detection of lysosomal membrane permeabilization by lysosomal galectin puncta assay

Lysosomal membrane permeabilization (LMP) contributes to tissue involution, degenerative diseases, and cancer therapy. Its investigation has, however, been hindered by the lack of sensitive methods. Here, we characterize and validate the detection of galectin puncta at leaky lysosomes as a highly sensitive and easily manageable assay for LMP. LGALS1/galectin-1 and LGALS3/galectin-3 are best suited for this purpose due to their widespread expression, rapid translocation to leaky lysosomes and availability of high-affinity antibodies. Galectin staining marks individual leaky lysosomes early during lysosomal cell death and is useful when defining whether LMP is a primary or secondary cause of cell death. This sensitive method also reveals that cells can survive limited LMP and confirms a rapid formation of autophagic structures at the site of galectin puncta. Importantly, galectin staining detects individual leaky lysosomes also in paraffin-embedded tissues allowing us to demonstrate LMP in tumor xenografts in mice treated with cationic amphiphilic drugs and to identify a subpopulation of lysosomes that initiates LMP in involuting mouse mammary gland. The use of ectopic fluorescent galectins renders the galectin puncta assay suitable for automated screening and visualization of LMP in live cells and animals. Thus, the lysosomal galectin puncta assay opens up new possibilities to study LMP in cell death and its role in other cellular processes such as autophagy, senescence, aging, and inflammation.     

Novel cellular defenses against iron and oxidation: ferritin and autophagocytosis preserve lysosomal stability in airway epithelium

Adsorbed to a variety of particles, iron may be carried to the lungs by inhalation thereby contributing to a number of inflammatory lung disorders. Redox-active iron is a potent catalyst of oxidative processes, but intracellularly it is bound primarily to ferritin in a non-reactive form and probably is catalytically active largely within the lysosomal compartment. Damage to the membranes of these organelles causes the release to the cytosol of a host of powerful hydrolytic enzymes, inducing apoptotic or necrotic cell death. The results of this study, using cultured BEAS-2B cells, which are adenovirus transformed human bronchial epithelial cells, and A549 cells, which have characteristics similar to type II alveolar epithelial cells, suggest that the varying abilities of different types of lung cells to resist oxidative stress may be due to differences in intralysosomal iron chelation. Cellular ferritin and iron were assayed by ELISA and atomic absorption, while plasma and lysosomal membrane stability were evaluated by the acridine orange uptake and trypan blue dye exclusion tests, respectively. Normally, and also after exposure to an iron complex, A549 cells contained significantly more ferritin (2.26 +/- 0.60 versus 0.63 +/- 0.33 ng/microg protein, P <0.001) and less iron (0.96 +/- 0.14 versus 1.48 +/- 0.21 ng/microg protein, P <0.05) than did BEAS-2B cells. Probably as a consequence, iron-exposed A549 cells displayed more stable lysosomes (P <0.05) and better survival (P <0.05) following oxidative stress. Following starvation-induced autophagocytosis, which also enhances resistance to oxidant stress, the A549 cells showed a significant reduction in ferritin, and the BEAS-2B cells did not. These results suggest that intralysosomal ferritin enhances lysosomal stability by iron-chelation, preventing Fenton-type chemistry. This notion was further supported by the finding that endocytosis of apoferritin, added to the medium, stabilized lysosomes (P <0.001 versus P <0.01) and increased survival (P <0.01 versus P <0.05) of iron-loaded A549 and BEAS-2B cells. Assuming that primary cell lines of the alveolar and bronchial epithelium behave in a similar manner as these respiratory cell lines, intrabronchial instillation of apoferritin-containing liposomes may in the future be a treatment for iron-dependent airway inflammatory processes.     

FV-429 induces autophagy blockage and lysosome-dependent cell death of T-cell malignancies via lysosomal dysregulation

It is widely accepted that lysosomes are essential for cell homeostasis, and autophagy plays an important role in tumor development. Here, we found FV-429, a synthetic flavonoid compound, inhibited autophagy flux, promoted autophagosomes accumulation, and inhibited lysosomal degradation in T-cell malignancies. These effects were likely to be achieved by lysosomal dysregulation. The destructive effects of FV-429 on lysosomes resulted in blockage of lysosome-associated membrane fusion, lysosomal membrane permeabilization (LMP), and cathepsin-mediated caspase-independent cell death (CICD). Moreover, we initially investigated the effects of autophagy inhibition by FV-429 on the therapeutic efficacy of chemotherapy and found that FV-429 sensitized cancer cells to chemotherapy agents. Our findings suggest that FV-429 could be a potential novel autophagy inhibitor with notable antitumor efficacy as a single agent.Subject terms: Haematological cancer, Macroautophagy, Pharmacology     

Ferritin-stimulated lipid peroxidation,lysosomal leak,and macroautophagy promote lysosomal “metastability” in primary hepatocytes determining in vitro cell survival

Several pathologies are associated with elevated levels of serum ferritin, for which growth inhibitory properties have been reported; however, the underlying mechanisms are still poorly defined. Previously we have described cytotoxic properties of isoferritins released from primary hepatocytes in vitro, which induce apoptosis in an iron and oxidative stress-dependent mode. Here we show that this ferritin species stimulates endosome clustering and giant endosome formation in primary hepatocytes accompanied by enhanced lysosomal membrane permeability (LMP). In parallel, protein modification by lipid peroxidation-derived 4-hydroxynonenal (HNE) is strongly promoted by ferritin, the HNE-modified proteins (HNE-P) showing remarkable aggregation. Emphasizing the prooxidant context, GSH is rapidly depleted and the GSH/GSSG ratio is substantially declining in ferritin-treated cells. Furthermore, ferritin triggers a transient upregulation of macroautophagy which is abolished by iron chelation and apparently supports HNE-P clearance. Macroautophagy inhibition by 3-methyladenine strongly amplifies ferritin cytotoxicity in a time- and concentration-dependent mode, suggesting an important role of macroautophagy on cellular responses to ferritin endocytosis. Moreover, pointing at an involvement of lysosomal proteolysis, ferritin cytotoxicity and lysosome fragility are aggravated by the protease inhibitor leupeptin. In contrast, EGF which suppresses ferritin-induced cell death attenuates ferritin-mediated LMP. In conclusion, we propose that HNE-P accumulation, lysosome dysfunction, and macroautophagy stimulated by ferritin endocytosis provoke lysosomal “metastability” in primary hepatocytes which permits cell survival as long as in- and extrinsic determinants (e.g., antioxidant availability, damage repair, EGF signaling) keep the degree of lysosomal destabilization below cell death-inducing thresholds.     

On the Cytoprotective Role of Ferritin in Macrophages and its Ability to Enhance Lysosomal Stability

Macrophages have a great capacity to take up (eg. by endocytosis and phagocytosis) exogenous sources of iron which could potentially become cytotoxic, particularly following the intralysosomal formation of low-molecular weight, redox active iron, and under conditions of oxidative stress. Following autophago-cytosis of endogenous ferritin/apoferritin, these compounds may serve as chelators of such lysosomal iron and counteract the occurrence of iron-mediated intralysosomal oxidative reactions. Such redox-reactions have been shown to lead to destabilisation of lysosomal membranes and result in leakage of damaging lysosomal contents to the cytosol. In this study we have shown: (i) human monocyte-derived macrophages to accumulate ferritin in response to iron exposure; (ii) iron to destabilise macrophage secondary lysosomes when the cells are exposed to H₂O₂; and (iii) endocytosed apoferritin to act as a stabiliser of the acidic vacuolar compartment of iron-loaded macrophages. While the endogenous ferritin accumulation which was induced by iron exposure was not sufficient to protect cells from the damaging effects of H₂O₂, exogenously added apoferritin, as well as the potent iron chelator desferrioxamine, afforded significant protection. It is suggested that intralysosomal formation of haemosiderin, from partially degraded ferritin, is a protective strategy to suppress intralysosomal iron-catalysed redox reactions. However, under conditions of severe macrophage lysosomal iron-overload, induction of ferritin synthesis is not enough to completely prevent the enhanced cytotoxic effects of H₂O₂.     

Starvation-induced autophagocytosis paradoxically decreases the susceptibility to oxidative stress of the extremely oxidative stress-sensitive NIT insulinoma cells

Summary

Glucose and amino acid starvation of cells in culture generally enhances their sensitivity to oxidative stress. This is explained by compensatory autophagocytosis, which results in increased amounts of lysosomal low-molecular-weight, redox-active iron, due to the degradation of metallo-proteins, with a potential increase in iron-catalyzed, intralysosomal oxidative reactions. Such reactions diminish the stability of lysosomal membranes, with resultant leakage of hydrolytic enzymes into the cytosol and ensuing cellular degeneration, often of apoptotic type. However, starvation of NIT insulinoma cells, which are normally remarkably sensitive to oxidative stress, actually attenuated the sensitivity to such stress. We found that starved NIT cells rapidly synthesized ferritin. Moreover, ferritin was found to be autophagocytosed, and the lysosomes were stabilized, as assayed by the acridine orange relocation test. We hypothesize that compensatory autophagocytosis during starvation increases the cytosolic pool of redox-active iron, as a reflection of enhanced transportation of low-molecular-weight iron from autophagic lysosomes to the cytosol, resulting in ferritin induction. The newly formed ferritin would, in turn, become autophagocytosed and bind redox-active lysosomal iron in a non-redox-active form. We also suggest that the proposed mechanism may be a way for oxidative stress-sensitive cells to compensate partly for their failing capacity to degrade hydrogen peroxide before it leaks into the acidic vacuolar apparatus and induces intralysosomal oxidative stress. The insulin-producing beta cell may belong to this type of cells.     

Oxidative stress induces macroautophagy of amyloid β-protein and ensuing apoptosis

There is increasing evidence for the toxicity of intracellular amyloid β-protein (Aβ) to neurons and the involvement of lysosomes in this process in Alzheimer disease (AD). We have recently shown that oxidative stress, a recognized determinant of AD, enhances macroautophagy and leads to intralysosomal accumulation of Aβ in cultured neuroblastoma cells. We hypothesized that oxidative stress promotes AD by stimulating macroautophagy of Aβ that further may induce cell death by destabilizing lysosomal membranes. To investigate such possibility, we compared the effects of hyperoxia (40% ambient oxygen) in cultured HEK293 cells that were transfected with an empty vector (Vector), wild-type APP (APPwt), or Swedish mutant APP (APPswe). Exposure to hyperoxia for 5 days increased the number of cells with Aβ-containing lysosomes, as well as the number of apoptotic cells, compared to normoxic conditions. The rate of apoptosis in all three cell lines demonstrated dependence on intralysosomal Aβ content (Vector < APPwt < APPswe). Furthermore, the degree of apoptosis was positively correlated with lysosomal membrane permeabilization, whereas inhibitors of macroautophagy and lysosomal function decreased oxidant-induced apoptosis and diminished the differences in apoptotic response between different cell lines. These results suggest that oxidative stress can induce neuronal death through macroautophagy of Aβ and consequent lysosomal membrane permeabilization, which may help explain the mechanisms behind neuronal loss in AD.     

Concanavalin A/IFN-gamma triggers autophagy-related necrotic hepatocyte death through IRGM1-mediated lysosomal membrane disruption

Interferon-gamma (IFN-γ), a potent Th1 cytokine with multiple biological functions, can induce autophagy to enhance the clearance of the invading microorganism or cause cell death. We have reported that Concanavalin A (Con A) can cause autophagic cell death in hepatocytes and induce both T cell-dependent and -independent acute hepatitis in immunocompetent and immunodeficient mice, respectively. Although IFN-γ is known to enhance liver injury in Con A-induced hepatitis, its role in autophagy-related hepatocyte death is not clear. In this study we report that IFN-γ can enhance Con A-induced autophagic flux and cell death in hepatoma cell lines. A necrotic cell death with increased lysosomal membrane permeabilization (LMP) is observed in Con A-treated hepatoma cells in the presence of IFN-γ. Cathepsin B and L were released from lysosomes to cause cell death. Furthermore, IFN-γ induces immunity related GTPase family M member 1(IRGM1) translocation to lysosomes and prolongs its activity in Con A-treated hepatoma cells. Knockdown of IRGM1 inhibits the IFN-γ/Con A-induced LMP change and cell death. Furthermore, IFN-γ(-/-) mice are resistant to Con A-induced autophagy-associated necrotic hepatocyte death. We conclude that IFN-γ enhances Con A-induced autophagic flux and causes an IRGM1-dependent lysosome-mediated necrotic cell death in hepatocytes.     

Endogenous ferritin protects cells with iron-laden lysosomes against oxidative stress

Previous studies have shown that a variety of mammalian cell types, including macrophages, contain small amounts of redox-active iron in their lysosomes. Increases in the level of this iron pool predispose the cell to oxidative stress. Limiting the availability of intralysosomal redox-active iron could therefore represent potential cytoprotection for cells under oxidative stress.

In the present study we have shown that an initial 6 h exposure of J774 macrophages to 30 μM iron, added to the culture medium as FeCl₃, increased the lysosomal iron content and their sensitivity to H₂O₂-induced (0.25 mM for 30 min) oxidative stress. Over time (24-72 h), however, the cells were desensitized to the cytotoxic effects of H₂O₂; most likely as a consequence of both lysosomal iron exocytosis and of ferritin synthesis (demonstrated by atomic absorption spectrophotometry, autometallography, and immunohistochemistry). When the cells were exposed to a second dose of iron, their lysosomal content of iron increased again but the cells became no further sensitized to the cytotoxic effects of H₂O₂. Using the lysosomotropic weak base, acridine orange, we demonstrated that after the second exposure to iron and H₂O₂, lysosomes remained intact and were no different from control cells which were exposed to H₂O₂ but not iron.

These data suggest that the initial induction of ferritin synthesis leads to enrichment of lysosomes with ferritin via autophagocytosis. This limits the redox-availability of intralysosomal iron and, in turn, decreases the cells' sensitivity to oxidative stress. These in vitro observations could also explain why cells under pathological conditions, such as haemochromatosis, are apparently able to withstand high iron concentrations for some time in vivo.     

Autophagy as a second level protective process in conferring resistance to environmentally-induced oxidative stress

This conceptual paper addresses the role of lysosomal autophagy in cellular defense against environmentally-induced oxidative stress using a marine mollusc (the blue mussel) as an experimental model. It is proposed that augmented autophagic removal of oxidatively damaged organelles and proteins provides a second level or tier of defense against oxidative stress. Age pigment or lipofuscin is a product of oxidative attack on proteins and lipids and can accumulate in lysosomes, where it may generate further reactive oxygen species (ROS) and inhibit lysosomal function, resulting in autophagic failure. The previously observed protective role of augmented autophagy, induced by nutritional deprivation, against oxidative stress can be explained by this model, where autophagy boosts "cellular housekeeping" through enhanced removal of ROS-damaged proteins and organelles minimizing formation of potentially harmful stress/age pigment, and has been proposed as an anti-aging mechanism. Finally, the probable low level triggering of autophagy in mussels by fluctuating environmental regimes is considered as a potential protective mechanism that will contribute to resistance to environmentally induced oxidative stress. It is further conjectured that organisms making up functional ecological assemblages (communities) in fluctuating environments, where upregulation of autophagy should provide a selective advantage, may be pre-selected to be tolerant of pollutant-induced oxidative stress.