Attenuation of iron-binding proteins in ARPE-19 cells reduces their resistance to oxidative stress.

PURPOSE: Oxidative stress-related damage to retinal pigment epithelial (RPE) cells is an important feature in the development of age-related macular degeneration. Iron-catalysed intralysosomal production of hydroxyl radicals is considered a major pathogenic factor, leading to lipofuscin formation with ensuing depressed cellular autophagic capacity, lysosomal membrane permeabilization and apoptosis. Previously, we have shown that cultured immortalized human RPE (ARPE-19) cells are extremely resistant to exposure to bolus doses of hydrogen peroxide and contain considerable amounts of the iron-binding proteins metallothionein (MT), heat-shock protein 70 (HSP70) and ferritin (FT). According to previous findings, autophagy of these proteins depresses lysosomal redox-active iron. The aim of this study was to investigate whether up- or downregulation of these proteins would affect the resistance of ARPE-19 cells to oxidative stress. METHODS: The sensitivity of ARPE-19 cells to H2 O2 exposure was tested following upregulation of MT, HSP70 and/or FT by pretreatment with ZnSO4 , heat shock or FeCl3 , as well as siRNA-mediated downregulation of the same proteins. RESULTS: Upregulation of MT, HSP70 and FT did not improve survival following exposure to H2 O2 . This was interpreted as existence of an already maximal protection. Combined siRNA-mediated attenuation of both FT chains (H and L), or simultaneous downregulation of all three proteins, made the cells significantly more susceptible to oxidative stress confirming the importance of iron-binding proteins. CONCLUSION: The findings support our hypothesis that the oxidative stress resistance exhibited by RPE cells may be explained by a high autophagic influx of iron-binding proteins that would keep levels of redox-active lysosomal iron low.

Increased levels of neutrophil extracellular trap remnants in the circulation of patients with small vessel vasculitis, but an inverse correlation to anti-neutrophil cytoplasmic antibodies during remission.

OBJECTIVES: Neutrophil extracellular traps (NETs) have been visualized at the site of ANCA-associated vasculitis (AAV) lesions. Increased levels of NET remnants in the circulation have been reported in some AAV patients with active disease. The aim of the present study was to analyse NET remnants in a larger cohort of AAV patients with varying degrees of disease activity and to elucidate possible factors responsible for remnant variation. METHODS: Levels of NET remnants in the circulation of healthy controls (HCs; n = 31) and AAV patients (n = 93) were determined with ELISA. NET remnants were then correlated with ANCA levels, spontaneous and induced cell death (NETosis/necrosis) in vitro, neutrophil count and corticosteroid therapy. RESULTS: Patients with active disease showed higher levels of circulating NET remnants compared with patients in remission (P = 0.026) and HCs (P = 0.006). From patients sampled during both remission and active disease, we found increased levels during active disease (P = 0.0010). In remission, ANCA-negative patients had higher levels of NET remnants than ANCA-positive patients and a negative correlation was observed between NET remnants and PR3-ANCA (rs = -0.287, P = 0.048). NET remnants correlated with neutrophil count in HCs (rs = 0.503, P = 0.014) but not in patients during remission. Neutrophils from patients showed enhanced spontaneous cell death (P = 0.043). CONCLUSION: We found increased levels of circulating NET remnants in patients with active AAV. Furthermore, AAV patients exhibited an increased propensity for spontaneous cell death. NET remnant levels seem to be positively related to disease activity and neutrophil count, but inversely related to ANCA at least during remission.

The neutrophil serine protease PR3 induces shape change of platelets via the Rho/Rho kinase and Ca(2+) signaling pathways.

INTRODUCTION: Proteinase 3 (PR3) is released from neutrophil azurophilic granules and exerts complex effects on the inflammatory process. PR3 catalyzes the degradation of a number of macromolecules, but the consequences on blood cells are less well defined. In the present study, the effect of PR3 on human platelets was thoroughly investigated. METHODS: The experiments were performed on washed platelets freshly isolated from blood donated by healthy human volunteers. Platelets shape change and aggregation was measured on a Chrono-Log aggregometer. The phosphorylated form of MYPT1 was visualized by immunostaining. Platelet activation was further evaluated by flow cytometry. RESULTS: PR3 induced platelet shape change but not aggregation. Flow cytometry analysis showed that PR3 induced no P-selectin expression or binding of fibrinogen to the platelets, and it did not change the activation in response to PAR1- or PAR4-activating peptides or to thrombin. Furthermore, Fura-2 measurement and immuno-blotting analysis, respectively, revealed that PR3 stimulated small intracellular Ca(2+) mobilization and Thr696-specific phosphorylation of the myosin phosphatase target subunit 1 (MYPT1). Separate treatment of platelets with the Rho/Rho kinase inhibitor Y-27632 and the intracellular Ca(2+) chelator BAPTA/AM reduced the shape change induced by PR3 whereas concurrent treatment completely inhibited it. CONCLUSION: The data shows that the neutrophil protease PR3 is a direct modulator of human platelets and causes shape change through activation of the Rho/Rho kinase and Ca(2+) signaling pathways. This finding highlights an additional mechanism in the complex interplay between neutrophils and platelets.

Lysosomal iron, iron chelation, and cell death.

SIGNIFICANCE: Lysosomes are acidic organelles containing more than fifty hydrolases that provide for the degradation of intracellular and endocytosed materials by autophagy and heterophagy, respectively. They digest a variety of macromolecules, as well as all organelles, and their integrity is crucial. As a result of the degradation of iron-containing macromolecules (e.g., ferritin and mitochondrial components) or endocytosed erythrocytes (by macrophages), lysosomes can accumulate large amounts of iron. This iron occurs often as Fe(II) due to the acidic and reducing lysosomal environment. Fe(II) is known to catalyze Fenton reactions, yielding extremely reactive hydroxyl radicals that may jeopardize lysosomal membrane integrity during oxidative stress. This results in the release of hydrolases and redox-active iron into the cytosol with ensuing damage or cell death. Lysosomes play key roles not only in apoptosis and necrosis but also in neurodegeneration, aging, and atherosclerosis. RECENT ADVANCES: The damaging effect of intralysosomal iron can be hampered by endogenous or exogenous iron chelators that enter the lysosomal compartment by membrane permeation, endocytosis, or autophagy. CRITICAL ISSUES: Cellular sensitivity to oxidative stress is enhanced by lysosomal redox-active iron or by lysosomal-targeted copper chelators binding copper (from degradation of copper-containing macromolecules) in redox-active complexes. Probably due to higher copper levels, lysosomes of malignant cells may be specifically sensitized by such chelators. FUTURE DIRECTIONS: By increasing lysosomal redox-active iron or exposing cells to lysosomal-targeted copper chelators, it should be possible to enhance the sensitivity of cancer cells to radiation-induced oxidative stress or treatment with cytostatics that induce such stress.

Resistance to genotoxic stresses in Arctica islandica, the longest living noncolonial animal: is extreme longevity associated with a multistress resistance phenotype?

Bivalve molluscs are newly discovered models of successful aging. Here, we test the hypothesis that extremely long-lived bivalves are not uniquely resistant to oxidative stressors (eg, tert-butyl hydroperoxide, as demonstrated in previous studies) but exhibit a multistress resistance phenotype. We contrasted resistance (in terms of organismal mortality) to genotoxic stresses (including topoisomerase inhibitors, agents that cross-link DNA or impair genomic integrity through DNA alkylation or methylation) and to mitochondrial oxidative stressors in three bivalve mollusc species with dramatically differing life spans: Arctica islandica (ocean quahog), Mercenaria mercenaria (northern quahog), and the Atlantic bay scallop, Argopecten irradians irradians (maximum species life spans: >500, >100, and ~2 years, respectively). With all stressors, the short-lived A i irradians were significantly less resistant than the two longer lived species. Arctica islandica were consistently more resistant than M mercenaria to mortality induced by oxidative stressors as well as DNA methylating agent nitrogen mustard and the DNA alkylating agent methyl methanesulfonate. The same trend was not observed for genotoxic agents that act through cross-linking DNA. In contrast, M mercenaria tended to be more resistant to epirubicin and genotoxic stressors, which cause DNA damage by inhibiting topoisomerases. To our knowledge, this is the first study comparing resistance to genotoxic stressors in bivalve mollusc species with disparate longevities. In line with previous studies of comparative stress resistance and longevity, our data extends, at least in part, the evidence for the hypothesis that an association exists between longevity and a general resistance to multiplex stressors, not solely oxidative stress. This work also provides justification for further investigation into the interspecies differences in stress response signatures induced by a diverse array of stressors in short-lived and long-lived bivalves, including pharmacological agents that elicit endoplasmic reticulum stress and cellular stress caused by activation of innate immunity.

Autophagy of iron-binding proteins may contribute to the oxidative stress resistance of ARPE-19 cells.

The objective of this study was to elucidate possible reasons for the remarkable resistance of human retinal pigment epithelial (RPE) cells to oxidative stress. Much oxidative damage is due to hydrogen peroxide meeting redox-active iron in the acidic and reducing lysosomal environment, resulting in the production of toxic hydroxyl radicals that may oxidize intralysosomal content, leading to lipofuscin (LF) formation or, if more extensive, to permeabilization of lysosomal membranes. Formation of LF is a risk factor for age-related macular degeneration (AMD) and known to jeopardize normal autophagic rejuvenation of vital cellular biomolecules. Lysosomal membrane permeabilization causes release of lysosomal content (redox-active iron, lytic enzymes), which may then cause cell death. Total cellular and lysosomal low-mass iron of cultured, immortalized human RPE (ARPE-19) cells was compared to that of another professional scavenger cell line, J774, using atomic absorption spectroscopy and the cytochemical sulfide-silver method (SSM). It was found that both cell lines contained comparable levels of total as well as intralysosomal iron, suggesting that the latter is mainly kept in a non-redox-active state in ARPE-19 cells. Basal levels and capacity for upregulation of the iron-binding proteins ferritin, metallothionein and heat shock protein 70 were tested in both cell lines using immunoblotting. Compared to J774 cells, ARPE-19 cells were found to contain very high basal levels of all these proteins, which could be even further upregulated following appropriate stimulation. These findings suggest that a high basal expression of iron-binding stress proteins, which during their normal autophagic turnover in lysosomes may temporarily bind iron prior to their degradation, could contribute to the unusual oxidative stress-resistance of ARPE-19 cells. A high steady state influx of such proteins into lysosomes would keep the level of lysosomal redox-active iron permanently low. This, in turn, should delay intralysosomal accumulation of LF in RPE cells, which is known to reduce autophagic turnover as well as uptake and degradation of worn out photoreceptor tips. This may explain why severe LF accumulation and AMD normally do not develop until fairly late in life, in spite of RPE cells being continuously exposed to high levels of oxygen and light, as well as large amounts of lipid-rich material.

Effects of MnDPDP and ICRF-187 on Doxorubicin-Induced Cardiotoxicity and Anticancer Activity.

Oxidative stress participates in doxorubicin (Dx)-induced cardiotoxicity. The metal complex MnDPDP and its metabolite MnPLED possess SOD-mimetic activity, DPDP and PLED have, in addition, high affinity for iron. Mice were injected intravenously with MnDPDP, DPDP, or dexrazoxane (ICRF-187). Thirty minutes later, mice were killed, the left atria were hung in organ baths and electrically stimulated, saline or Dx was added, and the contractility was measured for 60 minutes. In parallel experiments, 10 µM MnDPDP or MnPLED was added directly into the organ bath. The effect of MnDPDP on antitumor activity of Dx against two human tumor xenografts (MX-1 and A2780) was investigated. The in vitro cytotoxic activity was studied by co-incubating A2780 cells with MnDPDP, DPDP, and/or Dx. Dx caused a marked reduction in contractile force. In vivo treatment with MnDPDP and ICRF-187 attenuated the negative effect of Dx. When added directly into the bath, MnDPDP did not protect, whereas MnPLED attenuated the Dx effect by approximately 50%. MnDPDP or ICRF-187 did not interfere negatively with the anti-tumor activity of Dx, either in vivo or in vitro. Micromolar concentrations of DPDP but not MnDPDP displayed an in vitro cytotoxic activity against A2780 cells. The present results show that MnDPDP, after being metabolized to MnPLED, protects against acute Dx cardiotoxicity. Both in vivo and in vitro experiments show that cardioprotection takes place without interfering negatively with the anticancer activity of Dx. Furthermore, the results suggest that the previously described cytotoxic in vivo activity of MnDPDP is an inherent property of DPDP.

Superior therapeutic index of calmangafodipir in comparison to mangafodipir as a chemotherapy adjunct.

Mangafodipir is a magnetic resonance imaging contrast agent with manganese superoxide dismutase (MnSOD) mimetic activity. The MnSOD mimetic activity protects healthy cells against oxidative stress-induced detrimental effects, e.g., myelosuppressive effects of chemotherapy drugs. The contrast property depends on in vivo dissociation of Mn(2+) from mangafodipir-about 80% dissociates after injection. The SOD mimetic activity, however, depends on the intact Mn complex. Complexed Mn(2+) is readily excreted in the urine, whereas dissociated Mn(2+) is excreted slowly via the biliary route. Mn is an essential but also a potentially neurotoxic metal. For more frequent therapeutic use, neurotoxicity due to Mn accumulation in the brain may represent a serious problem. Replacement of 4/5 of Mn(2+) in mangafodipir with Ca(2+) (resulting in calmangafodipir) stabilizes it from releasing Mn(2+) after administration, which roughly doubles renal excretion of Mn. A considerable part of Mn(2+) release from mangafodipir is governed by the presence of a limited amount of plasma zinc (Zn(2+)). Zn(2+) has roughly 10(3) and 10(9) times higher affinity than Mn(2+) and Ca(2+), respectively, for fodipir. Replacement of 80% of Mn(2+) with Ca(2+) is enough for binding a considerable amount of the readily available plasma Zn(2+), resulting in considerably less Mn(2+) release and retention in the brain and other organs. At equivalent Mn(2+) doses, calmangafodipir was significantly more efficacious than mangafodipir to protect BALB/c mice against myelosuppressive effects of the chemotherapy drug oxaliplatin. Calmangafodipir did not interfere negatively with the antitumor activity of oxaliplatin in CT26 tumor-bearing syngenic BALB/c mice, contrary calmangafodipir increased the antitumor activity.

The role of lysosomes in iron metabolism and recycling.

Iron is the most abundant transition metal in the earth's crust. It cycles easily between ferric (oxidized; Fe(III)) and ferrous (reduced; Fe(II)) and readily forms complexes with oxygen, making this metal a central player in respiration and related redox processes. However, 'loose' iron, not within heme or iron-sulfur cluster proteins, can be destructively redox-active, causing damage to almost all cellular components, killing both cells and organisms. This may explain why iron is so carefully handled by aerobic organisms. Iron uptake from the environment is carefully limited and carried out by specialized iron transport mechanisms. One reason that iron uptake is tightly controlled is that most organisms and cells cannot efficiently excrete excess iron. When even small amounts of intracellular free iron occur, most of it is safely stored in a non-redox-active form in ferritins. Within nucleated cells, iron is constantly being recycled from aged iron-rich organelles such as mitochondria and used for construction of new organelles. Much of this recycling occurs within the lysosome, an acidic digestive organelle. Because of this, most lysosomes contain relatively large amounts of redox-active iron and are therefore unusually susceptible to oxidant-mediated destabilization or rupture. In many cell types, iron transit through the lysosomal compartment can be remarkably brisk. However, conditions adversely affecting lysosomal iron handling (or oxidant stress) can contribute to a variety of acute and chronic diseases. These considerations make normal and abnormal lysosomal handling of iron central to the understanding and, perhaps, therapy of a wide range of diseases.

Ascorbate and endocytosed Motexafin gadolinium induce lysosomal rupture.

Motexafin gadolinium (MGd) sensitizes malignant cells to ionizing radiation, although the underlying mechanisms for uptake and sensitization are both unclear. Here we show that MGd is endocytosed by the clathrin-dependent pathway with ensuing lysosomal membrane permeabilization, most likely via formation of reactive oxygen species involving redox-active metabolites, such as ascorbate. We propose that subsequent apoptosis is a synergistic effect of irradiation and high MGd concentrations in malignant cells due to their pronounced endocytic activity. The results provide novel insights into the mode of action of this promising anti-cancer drug, which is currently under clinical trials.

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.

Chelation of lysosomal iron protects against ionizing radiation.

Ionizing radiation causes DNA damage and consequent apoptosis, mainly due to the production of hydroxyl radicals (HO•) that follows radiolytic splitting of water. However, superoxide (O2•-) and H2O2 also form and induce oxidative stress with resulting LMP (lysosomal membrane permeabilization) arising from iron-catalysed oxidative events. The latter will contribute significantly to radiation-induced cell death and its degree largely depends on the quantities of lysosomal redox-active iron present as a consequence of autophagy and endocytosis of iron-rich compounds. Therefore radiation sensitivity might be depressed by lysosome-targeted iron chelators. In the present study, we have shown that cells in culture are significantly protected from ionizing radiation damage if initially exposed to the lipophilic iron chelator SIH (salicylaldehyde isonicotinoyl hydrazone), and that this effect is based on SIH-dependent lysosomal stabilization against oxidative stress. According to its dose-response-modifying effect, SIH is a most powerful radioprotector and a promising candidate for clinical application, mainly to reduce the radiation sensitivity of normal tissue. We propose, as an example, that inhalation of SIH before each irradiation session by patients undergoing treatment for lung malignancies would protect normally aerated lung tissue against life-threatening pulmonary fibrosis, whereas the sensitivity of malignant lung tumours, which usually are non-aerated, will not be affected by inhaled SIH.

What does the commonly used DCF test for oxidative stress really show?

H(2)DCF-DA (dihydrodichlorofluorescein diacetate) is widely used to evaluate 'cellular oxidative stress'. After passing through the plasma membrane, this lipophilic and non-fluorescent compound is de-esterified to a hydrophilic alcohol [H(2)DCF (dihydrodichlorofluorescein)] that may be oxidized to fluorescent DCF (2',7'-dichlorofluorescein) by a process usually considered to involve ROS (reactive oxygen species). It is, however, not always recognized that, being a hydrophilic molecule, H(2)DCF does not cross membranes, except for the outer fenestrated mitochondrial ones. It is also not generally realized that oxidation of H(2)DCF is dependent either on Fenton-type reactions or on unspecific enzymatic oxidation by cytochrome c, for neither superoxide, nor H(2)O(2), directly oxidizes H(2)DCF. Consequently, oxidation of H(2)DCF requires the presence of either cytochrome c or of both redox-active transition metals and H(2)O(2). Redox-active metals exist mainly within lysosomes, whereas cytochrome c resides bound to the outer side of the inner mitochondrial membrane. Following exposure to H(2)DCF-DA, weak mitochondrial fluorescence was found in both the oxidation-resistant ARPE-19 cells and the much more sensitive J774 cells. This fluorescence was only marginally enhanced following short exposure to H(2)O(2), showing that by itself it is unable to oxidize H(2)DCF. Cells that were either exposed to the lysosomotropic detergent MSDH (O-methylserine dodecylamide hydrochloride), exposed to prolonged oxidative stress, or spontaneously apoptotic showed lysosomal permeabilization and strong DCF-induced fluorescence. The results suggest that DCF-dependent fluorescence largely reflects relocation to the cytosol of lysosomal iron and/or mitochondrial cytochrome c.

Redox activity within the lysosomal compartment: implications for aging and apoptosis.

The lysosome is a redox-active compartment containing low-mass iron and copper liberated by autophagic degradation of metalloproteins. The acidic milieu and high concentration of thiols within lysosomes will keep iron in a reduced (ferrous) state, which can react with endogenous or exogenous hydrogen peroxide. Consequent intralysosomal Fenton reactions may give rise to the formation of lipofuscin or "age pigment" that accumulates in long-lived postmitotic cells that cannot dilute it by division. Extensive accumulation of lipofuscin seems to hinder normal autophagy and may be an important factor behind aging and age-related pathologies. Enhanced oxidative stress causes lysosomal membrane permeabilization, with ensuing relocation to the cytosol of iron and lysosomal hydrolytic enzymes, with resulting apoptosis or necrosis. Lysosomal copper is normally not redox active because it will form non-redox-active complexes with various thiols. However, if cells are exposed to lysosomotropic chelators that do not bind all the copper coordinates, highly redox-active complexes may form, with ensuing extensive lysosomal Fenton-type reactions and loss of lysosomal stability. Because many malignancies seem to have increased amounts of copper-containing macromolecules that are turned over by autophagy, it is conceivable that lysosomotropic copper chelators may be used in the future in ROS-based anticancer therapies.

Mitochondrial turnover and aging of long-lived postmitotic cells: the mitochondrial-lysosomal axis theory of aging.

It is now generally accepted that aging and eventual death of multicellular organisms is to a large extent related to macromolecular damage by mitochondrially produced reactive oxygen species, mostly affecting long-lived postmitotic cells, such as neurons and cardiac myocytes. These cells are rarely or not at all replaced during life and can be as old as the whole organism. The inherent inability of autophagy and other cellular-degradation mechanisms to remove damaged structures completely results in the progressive accumulation of garbage, including cytosolic protein aggregates, defective mitochondria, and lipofuscin, an intralysosomal indigestible material. In this review, we stress the importance of crosstalk between mitochondria and lysosomes in aging. The slow accumulation of lipofuscin within lysosomes seems to depress autophagy, resulting in reduced turnover of effective mitochondria. The latter not only are functionally deficient but also produce increased amounts of reactive oxygen species, prompting lipofuscinogenesis. Moreover, defective and enlarged mitochondria are poorly autophagocytosed and constitute a growing population of badly functioning organelles that do not fuse and exchange their contents with normal mitochondria. The progress of these changes seems to result in enhanced oxidative stress, decreased ATP production, and collapse of the cellular catabolic machinery, which eventually is incompatible with survival.

ARPE-19 retinal pigment epithelial cells are highly resistant to oxidative stress and exercise strict control over their lysosomal redox-active iron.

Normal retinal pigment epithelial (RPE) cells are postmitotic, long-lived and basically not replaced. Daily, they phagocytose substantial amounts of lipid-rich material (photoreceptor outer segment discs), and they do so in the most oxygenated part of the body-the retina. One would imagine that this state of affairs should be associated with a rapid formation of the age pigment lipofuscin (LF). However, LF accumulation is slow and reaches significant amounts only late in life when, if substantial, it often coincides with or causes age-related macular degeneration. LF formation occurs inside the lysosomal compartment as a result of iron-catalyzed peroxidation and polymerization. This process requires phagocytosed or autophagocytosed material under degradation, but also the presence of redox-active low mass iron and hydrogen peroxide. To gain some information on how RPE cells are able to evade LF formation, we investigated the response of immortalized human RPE cells (ARPE-19) to oxidative stress with/without the protection of a strong iron-chelator. The cells were found to be extremely resistant to hydrogen peroxide-induced lysosomal rupture and ensuing cell death. This marked resistance to oxidative stress was not explained by enhanced degradation of hydrogen peroxide, but to a certain extent further increased by the potent lipophilic iron chelator SIH. The cells were also able to survive, and even replicate, at high concentrations of SIH and showed a high degree of basal autophagic flux. We hypothesize that RPE cells have a highly developed capacity to keep lysosomal iron in a nonredox-active form, perhaps by pronounced autophagy of iron-binding proteins in combination with an ability to rapidly relocate low mass iron from the lysosomal compartment.

Autophagy of HSP70 and chelation of lysosomal iron in a non-redox-active form.

Lysosomes contain most of the cell's supply of labile iron, which makes them sensitive to oxidative stress. To keep lysosomal labile iron at a minimum, a cellular strategy might be to autophagocytose iron binding proteins that temporarily would chelate iron in a non-redox-active form. Previously we have shown that autophagy of metallothioneins, as well as of non-Fe-saturated ferritin, meets this goal. Here we add another stress-regulated protein to the list, namely HSP70.

The type IV mucolipidosis-associated protein TRPML1 is an endolysosomal iron release channel.

TRPML1 (mucolipin 1, also known as MCOLN1) is predicted to be an intracellular late endosomal and lysosomal ion channel protein that belongs to the mucolipin subfamily of transient receptor potential (TRP) proteins. Mutations in the human TRPML1 gene cause mucolipidosis type IV disease (ML4). ML4 patients have motor impairment, mental retardation, retinal degeneration and iron-deficiency anaemia. Because aberrant iron metabolism may cause neural and retinal degeneration, it may be a primary cause of ML4 phenotypes. In most mammalian cells, release of iron from endosomes and lysosomes after iron uptake by endocytosis of Fe(3+)-bound transferrin receptors, or after lysosomal degradation of ferritin-iron complexes and autophagic ingestion of iron-containing macromolecules, is the chief source of cellular iron. The divalent metal transporter protein DMT1 (also known as SLC11A2) is the only endosomal Fe(2+) transporter known at present and it is highly expressed in erythroid precursors. Genetic studies, however, suggest the existence of a DMT1-independent endosomal and lysosomal Fe(2+) transport protein. By measuring radiolabelled iron uptake, by monitoring the levels of cytosolic and intralysosomal iron and by directly patch-clamping the late endosomal and lysosomal membrane, here we show that TRPML1 functions as a Fe(2+) permeable channel in late endosomes and lysosomes. ML4 mutations are shown to impair the ability of TRPML1 to permeate Fe(2+) at varying degrees, which correlate well with the disease severity. A comparison of TRPML1(-/- )ML4 and control human skin fibroblasts showed a reduction in cytosolic Fe(2+) levels, an increase in intralysosomal Fe(2+) levels and an accumulation of lipofuscin-like molecules in TRPML1(-/-) cells. We propose that TRPML1 mediates a mechanism by which Fe(2+) is released from late endosomes and lysosomes. Our results indicate that impaired iron transport may contribute to both haematological and degenerative symptoms of ML4 patients.

Lysosomes and oxidative stress in aging and apoptosis.

The lysosomal compartment consists of numerous acidic vesicles (pH approximately 4-5) that constantly fuse and divide. It receives a large number of hydrolases from the trans-Golgi network, while their substrates arrive from both the cell's outside (heterophagy) and inside (autophagy). Many macromolecules under degradation inside lysosomes contain iron that, when released in labile form, makes lysosomes sensitive to oxidative stress. The magnitude of generated lysosomal destabilization determines if reparative autophagy, apoptosis, or necrosis will follow. Apart from being an essential turnover process, autophagy is also a mechanism for cells to repair inflicted damage, and to survive temporary starvation. The inevitable diffusion of hydrogen peroxide into iron-rich lysosomes causes the slow oxidative formation of lipofuscin in long-lived postmitotic cells, where it finally occupies a substantial part of the volume of the lysosomal compartment. This seems to result in a misdirection of lysosomal enzymes away from autophagosomes, resulting in depressed autophagy and the accumulation of malfunctioning mitochondria and proteins with consequent cellular dysfunction. This scenario might put aging into the category of autophagy disorders.

Lysosomes in iron metabolism, ageing and apoptosis.

The lysosomal compartment is essential for a variety of cellular functions, including the normal turnover of most long-lived proteins and all organelles. The compartment consists of numerous acidic vesicles (pH approximately 4 to 5) that constantly fuse and divide. It receives a large number of hydrolases ( approximately 50) from the trans-Golgi network, and substrates from both the cells' outside (heterophagy) and inside (autophagy). Many macromolecules contain iron that gives rise to an iron-rich environment in lysosomes that recently have degraded such macromolecules. Iron-rich lysosomes are sensitive to oxidative stress, while 'resting' lysosomes, which have not recently participated in autophagic events, are not. The magnitude of oxidative stress determines the degree of lysosomal destabilization and, consequently, whether arrested growth, reparative autophagy, apoptosis, or necrosis will follow. Heterophagy is the first step in the process by which immunocompetent cells modify antigens and produce antibodies, while exocytosis of lysosomal enzymes may promote tumor invasion, angiogenesis, and metastasis. Apart from being an essential turnover process, autophagy is also a mechanism by which cells will be able to sustain temporary starvation and rid themselves of intracellular organisms that have invaded, although some pathogens have evolved mechanisms to prevent their destruction. Mutated lysosomal enzymes are the underlying cause of a number of lysosomal storage diseases involving the accumulation of materials that would be the substrate for the corresponding hydrolases, were they not defective. The normal, low-level diffusion of hydrogen peroxide into iron-rich lysosomes causes the slow formation of lipofuscin in long-lived postmitotic cells, where it occupies a substantial part of the lysosomal compartment at the end of the life span. This seems to result in the diversion of newly produced lysosomal enzymes away from autophagosomes, leading to the accumulation of malfunctioning mitochondria and proteins with consequent cellular dysfunction. If autophagy were a perfect turnover process, postmitotic ageing and several age-related neurodegenerative diseases would, perhaps, not take place.