Lysosomes in Iron Metabolism

8/8/2019 Lysosomes in Iron Metabolism

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REVIEW

Lysosomes in iron metabolism, ageing and apoptosis

Tino Kurz

Alexei Terman

Bertil Gustafsson Ulf T. Brunk

Accepted: 24 January 2008 / Published online: 8 February 2008 Springer-Verlag 2008

Abstract The lysosomal compartment is essential for avariety of cellular functions, including the normal turnoverof most long-lived proteins and all organelles. The com-partment consists of numerous acidic vesicles (pH * 4 to 5)that constantly fuse and divide. It receives a large number of hydrolases ( * 50) from the trans -Golgi network, and sub-strates from both the cells outside (heterophagy) and inside(autophagy). Many macromolecules contain iron that givesrise to an iron-rich environment in lysosomes that recentlyhave degraded such macromolecules. Iron-rich lysosomesare sensitive to oxidative stress, while resting lysosomes,which have not recently participated in autophagic events,are not. The magnitude of oxidative stress determines thedegree of lysosomal destabilization and, consequently,whether arrested growth, reparative autophagy, apoptosis,or necrosis will follow. Heterophagy is the rst step in theprocess by which immunocompetent cells modify antigensand produce antibodies, while exocytosis of lysosomalenzymes may promote tumor invasion, angiogenesis, andmetastasis. Apart from being an essential turnover process,autophagy is also a mechanism by which cells will be ableto sustain temporary starvation and rid themselves of

intracellular organisms that have invaded, although somepathogens have evolved mechanisms to prevent theirdestruction. Mutated lysosomal enzymes are the underlyingcause of a number of lysosomal storage diseases involvingthe accumulation of materials that would be the substratefor the corresponding hydrolases, were they not defective.The normal, low-level diffusion of hydrogen peroxide intoiron-rich lysosomes causes the slow formation of lipofuscinin long-lived postmitotic cells, where it occupies a sub-stantial part of the lysosomal compartment at the end of thelife span. This seems to result in the diversion of newlyproduced lysosomal enzymes away from autophagosomes,leading to the accumulation of malfunctioning mitochon-dria and proteins with consequent cellular dysfunction. If autophagy were a perfect turnover process, postmitoticageing and several age-related neurodegenerative diseaseswould, perhaps, not take place.

Keywords Ageing Autophagy Lipofuscin Lysosomes Mitochondria Oxidative stress

AbbreviationsAO Acridine orangeATG Autophagy-related genesCMA Chaperone-mediated autophagyDFO DesferrioxamineLDL Low density lipoproteinLIP Labile iron poolLMP Lysosomal membrane permeabilityMMP Mitochondrial membrane permeabilityMP Mannose-6-phosphateMPR Mannose-6-phosphate receptorMPT Mitochondrial permeability transitionMSDH o-Methylserine dodecylamide hydrochloride

T. Kurz U. T. Brunk ( & )

Divisions of Pharmacology, Faculty of Health Sciences,Linko ping University, Linko ping, Swedene-mail: [email protected]

A. TermanDivisions of Geriatric Medicine,Faculty of Health Sciences, Linko ping University,Linko ping, Sweden

B. GustafssonDepartment of Pathology and Cytology,University Hospital, Linko ping, Sweden

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ROS Reactive oxygen speciesRPE Retinal pigment epithelialSIH Salicylaldehyde isonicotinoyl hydrazoneSSM Sulde-silver methodTf TransferrinTfR Transferrin receptorTGN trans -Golgi network

Introduction

It was the discoveries made by Christian de Duve and hiscoworkers in the late 1950s that brought lysosomes towider notice. The group, based at the Catholic Universityin Lovain, Belgium, was at the time conducting studies of glucose-6-phosphatase (reviewed in de Duve 2005 ). As areference enzyme, they used unspecic acid phosphatase.One day, when they decided to reinvestigate a cellhomogenate after keeping it for a day or two in a fridge,they found much higher activity of this enzyme comparedto the initial measurement. In contrast, the activity of glucose-6-phosphatase had not changed. In this wayenzymatic latency, a phenomenon that allows an enzymeto appear more active over time, was discovered. Usinggradient sedimentation, a new compartment was foundwith a density close to that of mitochondria. Their ndingsstrongly suggested that acid phosphatase was localizedwithin cytosolic membrane-bound vesicles of limited sta-bility, while glucose-6-phosphatase was not. With the helpof cytochemists and electron microscopists, particularlyAlex Novikoff and his colleagues, it was later found that anumber of acidic hydrolases, capable of degrading mostcomplex organic molecules, such as proteins, carbohy-drates, lipids and nucleotides, exist together inside anorganelle that was named lysosome (lytic body). Lyso-somes were observed to be a heterogeneous group of vacuoles of different sizes, form, and density. In thetransmission electron microscope, some lysosomes wereseen to contain still recognizable extra- and/or intracellularmaterial under degradation, while others looked homoge-neous or contained electron-dense whorls and clumps.

Soon after its recognition, the lysosomal compartmentbecame thought of as kind of stomach of the cell, and itsimportance in heterophagy (or endocytosis, which includesphagocytosis and pinocytosis) and autophagy (self-eating)was recognized. Some lysosomes were viewed as storageplaces for waste products, such as the age-pigment lipo-fuscin, and considered inactive without any remaining lyticcapacity. Such structures were named residual bodies ortelolysosomes. Later the assumption of inactivity provedto be wrong, and such lysosomes are now recognized asintegral parts of the lysosomal compartment and known to

receive newly produced lysosomal enzymes by fusion withother lysosomes (Brunk and Ericsson 1972a ).

Lysosomal enzymes are produced in the reticular net-work, matured in the cis -Golgi apparatus, and transportedfrom the trans -Golgi network (TGN) within tiny vesicles,sometimes called primary lysosomes, although they are notacidic and thus not true lysosomes. The latter fuse with lateendosomes and, maybe, also with autophagosomes (Dunn1990 ). Mature lysosomes are part of a dynamic system.They fuse and divide, allowing their content to be propa-gated throughout the compartment (Brunk 1973 ; Luzioet al. 2007 ). Since lipofuscin, or age pigment, is non-degradable, the transfer of active hydrolases to lipofuscin-loaded lysosomes, in a futile attempt to degrade the pig-ment, means a waste of enzymes. In the event thatlipofuscin-loaded lysosomes become plentiful, which cer-tainly is the case in senescent long-lived postmitotic cells,the latter may run short of functional lysosomal enzymes.Although the total amount of lysosomal enzymes may belarger than in young cells, much of it is misdirected. Insuch circumstances, the result is a shortfall in the supply of hydrolytic enzymes for useful purposes and, above all,inadequate autophagy with an accumulation of malfunc-tioning, enlarged mitochondria and aggregates of aberrantproteins (see Lipofuscin and postmitotic ageing ).

It was soon recognized that many previously knownstorage diseases, often with a serious outcome, are due tomutated and inactive lysosomal hydrolases. As a conse-quence, substrates cannot be degraded by these faultyenzymes, but rather accumulate inside the lysosomalcompartment. The analogy to lipofuscin accumulation insenescent long-lived postmitotic cells is obvious and, per-haps, we should look at ageing as a storage disorder wherelipofuscin is the material that accumulates, although forother reasons than defective lysosomal enzymes (seeLipofuscin and postmitotic ageing ). Recently it has beensuggested that both lysosomal storage diseases and ageingare associated with disturbed autophagy that causes theaccumulation of polyubiquitinated proteins and dysfunc-tional giant-type mitochondria (Kiselyov et al. 2007 ;Settembre et al. 2008 , 2008 ; Terman 1995 ; Terman andBrunk 2005a ). Consequently, senescence and lysosomalstorage diseases might, possibly, be considered autophagydisorders.

Although de Duve made an early suggestion that lyso-somes were suicide bags (de Duve 1959 ), they were for along time considered sturdy organelles, which would notrelease their potent mixture of hydrolases until cells werealready dead. Lysosomal enzymes would then be respon-sible for dissolving dead cells only (necrosis). This notionwas partly based on ultrastructural observations of seem-ingly intact lysosomes in devitalized cells. However, itlater turned out that at least the lysosomal enzyme acid

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phosphatase could leak from lysosomes that look normal inthe electron microscope (Brunk and Ericsson 1972b ), andthat lysosomes, contrary to general opinion at the time, aresensitive to stresses, especially to oxidants (Brunk andSvensson 1999 ; Kurz et al. 2004 , 2006 ; Persson et al. 2003 ;Yu et al. 2003b ). Many textbooks nevertheless still reectthe old opinion, and thus demonstrate the truth of Billings(18181885) statement: The trouble with people is notthat they dont know, but that they know so much that aintso (Billings 1874 ). The nding that the membranes of ratliver lysosomes contain a 37-fold higher specic content of a-tocopherol compared to other cellular membranes (Ruparet al. 1992 ) suggests that these membranes are in need of anti-oxidant protections, and is an indirect proof for thehypothesis that lysosomes normally are under the inuenceof substantial oxidative stress.

The rst reports of lysosomal destabilization or LMP(lysosomal membrane permeabilitycompare with MMP,mitochondrial membrane permeability), as an early eventin apoptosis appeared about fteen years ago. The dis-covery was a result of experiments on the induction of apoptosis in cultured cells by moderate oxidative stress.LMP was found to be a response to such stress, which wasthen apparently followed by MMP and classical apoptosis(reviewed in Brunk et al. 2001 ). Even earlier, however, itwas realized that a mitochondrial release of apoptogenicsubstances, such as cytochrome c, activates the caspasecascade and the internal pathway of apoptosis. Conse-quently, almost all researchers within the expanding eldof programmed cell death/apoptosis focused on the rolesof caspases and mitochondria in this process. Thehypothesis that lysosomes might play an important role in,at least, some forms of apoptosis had few advocatesamong most cell death researchers. During the last twodecades, a huge amount of knowledge has accumulated onapoptosis, but not until it was found that lysosomal cal-pain-like cysteine proteases truncate and activate the pro-apoptotic protein Bid (Cirman et al. 2004 ) did the role of lysosomes in apoptosis begin to become a somewhat morefashionable eld of investigation. Additional evidence forthe role of lysosomes in apoptosis was the nding that anendocytic uptake of the potent iron chelator desferriox-amine (DFO) gave rise to almost complete prevention(although only temporary see further in The stability of lysosomes under oxidative stress and their role in cellulariron metabolism ) of lysosomal rupture and ensuingapoptosis/necrosis when a number of different cell typeswere exposed to hydrogen peroxide (Antunes et al. 2001 ;Doulias et al. 2003 ; Yu et al. 2003b ). This nding gaverise to two essential conclusions: (1) the importance of redox-active iron in lysosomal stability at oxidative stress,and (2) that hydrogen peroxide per se is not particularlyharmful.

Even though the role of lysosomes in autophagy wasrecognized early (reviewed in Glaumann et al. 1981 ), thisphenomenon was long considered a murky one. This,however, radically changed when it was recently found thatautophagy is a much more regulated process than hithertobelieved, and under the inuence of more than a score of evolutionary well-preserved autophagy genes (ATG),which allow it to be selective and to take three differentforms: macro-autophagy, micro-autophagy, and chaperone-mediated autophagy (CMA) (reviewed in Cuervo et al.2005 ; Shintani and Klionsky 2004 ; Suzuki and Ohsumi2007 ; Yorimitsu and Klionsky 2005 ). More or less pro-nounced autophagy commonly occurs in apoptotic cells.Sometimes such autophagy is extensive, and apoptosis thendeviates from the classical form. This form of apoptosis hasbeen named autophagic cell death, or programmed celldeath type II, and is often considered to be caspase-inde-pendent (see Leaky lysosomes and apoptosis/necrosis ).

This review will focus on areas where lysosomes areimportant but not so often emphasized, and where theauthors themselves have some experience. They include:how lysosomes participate in cellular iron metabolism;lysosomal behavior under oxidative stress; how lysosomesand mitochondria interact to induce apoptosis; and, howlysosomes are involved in postmitotic ageing.

The lysosomal compartment and its main functions

The lysosomal compartment is indispensable for cellularlife and has several important functions. Lysosomes thusexist in all kinds of animal cells, except erythrocytes,which have a very specialized function and a minimalturnover of their constituents. The degradation of endocy-tosed or autophagocytosed materials takes place insidelysosomes that have an acidic (pH * 4 to 5) environment,which is maintained by ATP-dependent proton pumpspresent in the lysosomal membrane. Such pumps are alsopresent in the plasma membrane, especially in tumor cellswith a pronounced anaerobic glycolysis (the Warburgeffect) that results in a high production of lactic acid and aneed to export protons to avoid acidication of their owncytosol (Bartrons and Caro 2007 ; Semenza et al. 2001 ).

Following synthesis in the endoplasmic reticulum,lysosomal hydrolases are tagged with mannose-6-phos-phate (MP) in the cis -Golgi area and packaged intotransport vesicles (sometimes named primary lysosomesalthough they have a neutral pH) in the trans -Golgi net-work (TGN) with the help of MP receptors. The newlyproduced hydrolases are transported to slightly acidic (pHabout 6) late endosomes, which arise from early endosomescontaining endocytosed material. Here, the lysosomalhydrolases are freed from MP receptors and activated,

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while the MP receptors are re-circulated to the Golgiapparatus. The late endosomes then mature to lysosomesthat lack MP receptors, are rich in acid hydrolases, have apH of 45 and contain endocytosed material to be degra-ded. Lysosomes fuse with autophagosomes/endosomes toform hybrid organelles contaning material in the courseof degradation originating both from outside and inside thecell. Following completed degradation of the enclosedmaterial, lysosomes turn into resting ones, which in theelectron microscope look homogeneous and moderatelyelectron-dense. They are then ready for new rounds of fusion (Luzio et al. 2007 ). Indeed, pronounced fusion andssion activity is a characteristic of the lysosomal com-partment that allows lytic enzymes and other contents tospread through the compartment (see Fig. 1).

Following receptor-mediated endocytosis, the initiallyplasma membrane-bound receptors are often, but notalways, returned to the plasma membrane, while theligands are usually propagated further down the lysosomalcompartment. An exception is the tranferrin receptor andits transferrin ligand, which are both returned to the plasmamembrane, while the iron that is bound to transferrin isreleased by the acidity of the late endosomes (see Thestability of lysosomes under oxidative stress and their rolein cellular iron metabolism ).

The processing and presentation of antigens in immuno-competent cells is dependent on a form of endocytoticexocytotic activity, while autophagic degradation is of vitalimportance not only for the normal turnover of cellularconstituents, but also for the removal of damaged structuresand cytosolic microorganisms that have invaded the cell.Some cell types are able to exocytose lysosomal contentsor even intact lysosomes (secretory lysosomes) (reviewedin Luzio et al. 2007 ). It has been recognized that tumorcells often secrete lysosomal proteases, which, in combi-nation with acidication of their surroundings helps themto inltrate and spread (de Milito and Fais 2005 ; Lorenzoet al. 2000 ; Martinez-Zaguilan et al. 1993 ; Montcourrieret al. 1997 ; Podgorski and Sloane 2003 ; Rochefort et al.1990 ; Victor and Sloane 2007 ).

As pointed out above, lysosomes fuse with autophago-somes, or deliver part of their content (kiss-and-run), toform autophagolysosomes where a variety of organellesand proteins are degraded into simple components, whichin turn are reutilized by the anabolic machinery of the cellfollowing transport to the cytosol (reviewed in Klionsky2007 ; Terman et al. 2007 ).

From a physiological point of view, the lysosomalcompartment can be looked upon as a box, built of vacu-oles that constantly fuse and divide, that receives enzymes

Fig. 1 Autophagy can be subdivided into three forms: macro-, micro-and chaperone-mediated autophagy ( CMA ). In macroautophagy, acup-formed phagophore surrounds cytosolic organelles thereby cre-ating an autophagosome, which fuses with a lysosome or a lateendosome. Small portions of cytoplasm enter lysosomes throughinvagination of the membrane (microautophagy). In CMA, proteinstagged with the amino acid sequence KFERQ are bound by molecular

chaperones, e.g., Hsp73, and transported to lysosomes. The latterform from late endosomes from which they differ in that they do nothave mannose-6-phosphate receptors ( MPR ). Lysosomal hydrolasesare transported bound to MPRs in Golgi-derived vesicles to slightlyacidied late endosomes and, possibly, autophagosomes. There, theenzymes are released and the MPRs are recycled back to the Golginetwork


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from the TGN and substrates from either the outside or theinside of the cell. Following substrate degradation insideindividual lysosomes, the products diffuse or are activelytransported to the cytosol for reutilization.

Most of the mechanisms involved in the formation of theautophagic double membrane (the phagophore), the inclu-sion of materials to be degraded, and the fusion of autophagosomes and lysosomes was, as mentioned above,recently worked out as a result of the discovery of a largefamily of phylogenetically well preserved autophagy-rela-ted genes (ATG) (Klionsky 2007 ; Shintani and Klionsky2004 ; Suzuki and Ohsumi 2007 ; Yorimitsu and Klionsky2005 ). Autophagy represents one of the main pathways forthe turnover and reutilization of worn-out long-lived pro-teins and organelles and is a perfectly normal process.Interestingly, proteasomes, which also play an importantrole in the turnover of macromolecules, are themselvesdegraded by autophagy (Cuervo et al. 1995 ). The impli-cation of this is that hampered autophagy might result indefective proteasomes since they, in common with mito-chondria and other organelles, are then not properlyrenewed.

During periods of starvation, enhanced autophagy servesas a way for cells to survive by degrading less importantparts of themselves in order to produce ATP and buildingblocks for essential anabolic processes. Subsequent tocellular damage, reparative autophagy follows, in whichaltered and malfunctioning structures are replaced. Suchreparative autophagy is commonly seen following, forexample, ionizing irradiation, virus infection, and hypoxicor oxidative stress (Bergamini 2006 ; Kaushik and Cuervo2006 ).

The stability of lysosomes under oxidative stressand their role in cellular iron metabolism

As indicated above, apart from the operations of profes-sional scavengers with their high degree of endocytosis(heterophagy), autophagy is the main process that deliverssubstrates to the lysosomal compartment. Since manyautophagocytosed macromolecules contain iron (e.g., fer-ritin and mitochondrial electron-transport complexes), thelysosomal compartment becomes rich in this essential,although potentially hazardous, transition metal (Brun andBrunk 1970 ; Miyawaki 1965 ; Persson et al. 2003 ; Radiskyand Kaplan 1998 ; Roberts and Bomford 1988 ; Sakaidaet al. 1990 ; Schraufsta tter et al. 1988 ; Vaisman et al. 1997 ;Yu et al. 2003b ; Zdolsek et al. 1993 ). In this respect,lysosomes are unique (reviewed in Kurz et al. 2007 ). It isimportant to recognize that lysosomes that have recentlybeen engaged in an autophagic degradation of iron-richcompounds should contain high concentrations of iron,

while others which have been inactive for a while withrespect to such autophagy may contain only negligibleamounts of it (Kurz et al. 2007 ; Nilsson et al. 1997 ).Similarly, for some limited period of time, certain cells in apopulation may have been inactive altogether with respectto autophagy. If so, inside a particular cell we expect to ndlysosomes with a range of iron concentrations, and we alsoought to nd occasional cells with no, or almost no, lyso-somal iron. This hypothesis is supported by a line of evidence: (1) recent ndings indicate that the transport of released iron away from the lysosomal compartment is arapid process (T. Kurz et al., in preparation); (2) a pro-nounced variation is found in the stability of individuallysosomes against oxidative stress in single cells, and alsoin the total lysosomal population amongst cells (Nilssonet al. 1997 ); (3) the cytochemical sulde-silver method(SSM) for detection of loosely-bound iron shows sub-stantial variability with respect to amounts of lysosomaliron within as well as between cells. Figure 2 shows themainly lysosomal distribution of labile iron in J774 cellsusing the SSM. The heterogeneity with respect to lyso-somal iron is demonstrated by the relation between thedevelopment time and the number of positive lysosomes(for details see the legend to Fig. 2). The SSM demon-strates heavy metals, which normally means iron since it isthe only such metal that normally occurs in any signicantamount in most cells. Exceptions are pancreatic b -cells,prostatic epithelial cells and some neurons, all of which arerich in zinc (Zdolsek et al. 1993 ).

Taken together, these ndings would satisfactorilyexplain the difference in the sensitivity to oxidative stressfor lysosomes of individual cells as well as for a populationof cells, where always some cells survive even substantialstress, while others undergo apoptosis at a rather lowdegree of such stress. Such an explanation has been pointedout as essential to nd (Kroemer and Ja attela 2005 ). Therole of redox-active iron for the stability of lysosomesunder oxidative stress is further substantiated by the ndingthat the simultaneous exposure to hydrogen peroxide andthe potent lipophilic iron chelator salicylaldehyde isoni-cotinoyl hydrazone (SIH) almost fully prevents bothlysosomal rupture and apoptosis, as long as the stress is notoverwhelmingly strong. Likewise, the endocytotic uptakeof apo-ferritin and the autophagy of metallothioneins pro-tect lysosomes against oxidative stress. Conversely, theendocytotic uptake of an ironphosphate complex sensi-tizes to such stress (Fig. 3). The nding that hydrogenperoxide in concentrations that normally induce apoptosisor necrosis has no inuence on cells in the presence of SIH(apart from briey setting back their mitotic activity) tellsus that hydrogen peroxide per se is not particularly toxicbut rather must work in concert with iron in order todamage cells.


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Increased amounts of iron can accumulate in somelysosomes due to the accretion of iron-rich non-degradablematerials, such as lipofuscin and, especially, hemosiderinthat, perhaps, could be considered an unusually iron-richform of lipofuscin. Consequently, the sensitivity to oxida-tive stress of lysosomes loaded with lipofuscin orhemosiderin seems to be quite high (Seymour and Peters1978 ; Terman et al. 1999a ; Terman and Brunk 2004 ).

Non-dividing cells mainly rely on the turnover andreutilization of iron, while growing populations of cellsrequire its uptake from their surroundings. The uptake istightly controlled and accomplished by a complex series of events. Iron is carried in the circulation by transferrin (Tf)in a non-redox-active form. The TfFe complex binds tothe transferrin receptor (TfR), and the TfFeTfR complexis endocytosed and propagated into late endosomes. As aconsequence of the slightly acidic pH of late endosomes,

iron is released and transported to the cytosol by thedivalent metal transporter-1 (also known as Nramp 2 orSLC11A2). The TfTfR complex returns to the plasmamembrane, where transferrin is freed from the TfR andreleased to the circulation to pick up more iron. At neutralpH, FeTf binds to the TfR, while Tf does not. In thecytosol, low mass iron binds to specic iron regulatingproteins (IRPs), which in turn up-regulate ferritin anddown-regulate TfR, by interfering with the translation of their mRNA. By these mechanisms, cytosolic iron is kept

Fig. 2 Cytochemical demonstration of lysosomal iron in J774 cellsusing the high pH, high S 2

-sulphide-silver method ( SSM ). After

30 min of development ( a ), no positive lysosomes are seen. However,after 40 min of development ( b ) a few dark-stained positive (iron-containing) lysosomes are visible ( arrows ), indicating particularlyiron-rich lysosomes, while after 60 min of development ( c) a strongand widespread lysosomal-type pattern is found, indicating at leastsome iron in most lysosomes

Fig. 3 Labilization of lysosomes by hydrogen peroxide and itsmodulation by autophagocytosed metallothioneins and endocytosedapo-ferritin, desferrioxamine and a Fe phosphate complex. J774 cellswere exposed to hydrogen peroxide (initially 100 l M in PBS) for30 min (H 2 O2 ), to PBS only (Control), or to H 2 O2 in combinationwith 100 l M of the lipophilic iron-chelator SIHand then returnedto standard culture conditions. Some cells had previously beenexposed to the hydrophilic iron chelator desferrioxamine (1 mM) for3 h, to 1 l M apo-ferritin ( Apo-Ft ) for 4 h, or a Ferric-phosphatecomplex (30 l M) for 4 h, while other cells had been exposed toZnSO 4 for 12 h to induce the upregulation of metallothioneins andensuing autophagy of this protein. Note that apo-ferritin, desferriox-amine, SIH, and metallothioneins stabilize lysosomes, while the Fephosphate complex has the opposite effect. The gure is compiledfrom a number of previously published ones


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at the lowest possible level that allows synthesis of nec-essary iron-containing macromolecules (Kruszewski 2003 ).

Even though it is not known exactly how low mass ironis transported from lysosomes to the cytosol followingautophagic degradation of iron-containing macromole-cules, it may be assumed that mechanisms akin to thosethat transport iron from late endosomes are involved. It isalso not known whether iron that is bound to divalent metaltransporters is redox-active. However, natural processes areusually operated in a benign manner and, thus, it may beanticipated that, rst, cytosolic labile iron is in transit onlybriey before being taken care of in a safe way and, sec-ond, that while in transit its reactivity with respect toelectron transport is, at the least, restricted.

Recently, an alternative mechanism for the delivery of iron from late endosomes and lysosomes to mitochondriawas suggested. It involves a direct transport between thesetwo types of organelles following temporary close contact(kiss and run) (Zhang et al. 2005 ). For a schematic view of the involvement of lysosomes in iron metabolism, seeFig. 4.

Rapidly growing tumor cells have a high demand foriron and, therefore, iron chelators may be used to stop theirproliferation and induce apoptosis. Encouraging therapeu-tic results have been obtained in clinical phase III studiesusing iron chelators, which seem to have a selective effect

on tumor cells, while not harming normal ones that alsoneed iron, e.g., erythroblasts (Richardson 2002 ).

Some investigators have found support for the hypothesisthat when low mass iron is required, it is released directlyfrom ferritin within the cytosol (de Domenico et al. 2006 ;Liu et al. 2003 ; Takagi et al. 1998 ). Others favor an alter-native mechanism for iron release, namely autophagyfollowed by transport of released iron to the cytosol (Kidaneet al. 2006 ; Radisky and Kaplan 1998 ; Roberts and Bom-ford 1988 ; Sakaida et al. 1990 ; Tenopoulou et al. 2005 ;Vaisman et al. 1997 ; T. Kurz et al., in preparation). Thelatter hypothesis is supported by the ndings of manyelectron microscopists, who over the years have noticedferritin inside autophagosomes (Koorts and Viljoen 2007 ).Other arguments for the autophagy hypothesis is thatexposure of cells in culture to a panel of inhibitors tolysosomal enzymes prevents the release of iron from ferritin(Kidane et al. 2006 ), and the nding that exposure of cells tothe iron chelator DFO, which is endocytosed and trans-ported into the lysosomal compartment where it remainsnondegraded and acts as a sink for iron, rapidly results in amajor decrease of cytosolic labile (calcein-chelatable) iron(Tenopoulou et al. 2005 ). The latter nding supposedlyreects a rapid incorporation of cytosolic iron into ferritinor iron-containing macromolecules under construction.Such a rapid disappearance of calcein-chelatable iron from

Fig. 4 The role of lysosomes in cellular iron metabolism . In serum,iron is bound in a transferrin-iron complex that attaches to transferrinreceptors, which results in receptor-mediated endocytosis insideclathrin-coated vesicles. In the lowered pH of late endosomes, iron isreleased and transported to the cytoplasmic labile iron pool ( LIP ),while the transferrin and its receptor are recycled to the plasma

membrane. LIP iron is probably temporarily bound by low afnitychelators before it is either used in the synthesis of iron-containingproteins or stored in ferritin. It is unknown if LIP iron is redox-active.The LIP also receives iron from lysosomes following degradation of iron-containing proteins, e.g., ferritin and mitochondrial electrontransport complexes


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the cytosol is in agreement with the above notion thatcytosolic labile iron is in rapid transit and kept at the lowestpossible concentration in order to avoid Fenton-type reac-tions (Kruszewski 2003 ). If iron were liberated from ferritindirectly in the cytosol, and its concentration regulated by afeedback mechanism (de Domenico et al. 2006 ), one wouldexpect a preserved cytosolic concentration of chelatableiron for quite a while. However, whether autophagy and amechanism that involves direct iron-release from ferritin actin concert, or if one of them is dominantor even the onlyoneremains to be established.

As mentioned above, excessive iron may accumulatewithin lysosomes as a component of lipofuscin (reviewedin Brunk and Terman 2002 ; Jolly et al. 1995 ) or in the formof hemosiderin. The latter substance seems to represent aparticularly iron-rich form of lipofuscin (or ceroid, seebelow) composed of non-degradable polymerized alde-hyde-bridged ferritin residues. Because cells do not havethe capacity to rid themselves of intralysosomal iron that isbound to lipofuscin, it slowly accumulates over time, evenif the uptake of iron is effectively regulated. This accu-mulation is particularly evident in the reticulo-endothelialsystem, in hepatocytes, and in long-lived postmitotic cells,such as neurons and cardiac myocytes. It seems mostprobable that the sensitivity to oxidative stress is enhancedin such cells at the end of their lifespan.

As long as Fe(II) has at least one of its six coordinatesfree, it is capable of catalyzing a homolytic splitting of hydrogen peroxide resulting in the formation of the extre-mely reactive hydroxyl radical (Fenton reaction).

Fe2

H2O2!

Fe3

HO

OH

The favourable conditions for intralysomal Fenton reac-tions are a low internal pH ( * 4 to 5) and the presence of reducing equivalents, such as cysteine, ascorbic acid orglutathione (Baird et al. 2006 ; Pisoni et al. 1990 ; Schaferand Buettner 2000 ). At this pH, cysteine, as well as theother reducing agents, easily reduces iron(III) to iron(II).An additional reducing agent inside autophagolysosomesmay be the superoxide anion radical (O 2

-). Mitochondria

undergoing autophagic degradation probably continue toproduce these radicals for a while, perhaps even inincreased amounts, following disabling of the mitochon-drial electron transport complexes secondary to theproteolytic attack.

Hydrogen peroxide forms continuously in cells, mainlyby loss of electrons from the mitochondrial electron trans-porting chain, but also from a variety of oxido-reductasesthat operate by one-electron transfers. Some hydrogenperoxide may escape the effective cytosolic degradation bycatalase and glutathione peroxidase and diffuses into lyso-somes (Walton and Lewis 1916 ), which do not contain theseenzymes (those that are autophagocytosed are rapidly

degraded), to react with labile iron and form hydroxylradicals, or similarly reactive ferryl or perferryl compounds(reviewed in Eaton and Qian 2002 ) (see Fig. 5).

In normal conditions, with a low and steady state rate of autophagy, the result of the inevitable Fenton-type reac-tions inside lysosomes is a slow formation of lipofuscin(age-pigment) over time. Following enhanced autophagy,for example because of reparative efforts after injury, whenoften oxidative stress is present as well, a much more rapidformation of the pigment occurs. In such cases that areunrelated to normal ageing, the pigment is called ceroid,although both lipofuscin and ceroid basically have thesame composition and mode of origin, i.e., intralysosomaliron-catalyzed peroxidation of material under degradation(Brunk and Terman 2002 ; Terman and Brunk 1998 ).

When enhanced oxidative stress results in peroxidationnot only of the lysosomal contents but also of its mem-brane, lysosomes become leaky after a delay that isdependent on the magnitude of oxidative stress. Thishappens also if cells are exposed only briey to oxidativestress before being returned to standard culture conditionswithout further stress. Consequently, any satisfactoryhypothesis on the mechanisms behind oxidation-inducedlysosomal rupture must provide rm and distinct linksbetween the triggering events, which occur within minutes,and the ultimate leak that may be delayed by hours. Thislink seems to be lysosomal membrane peroxidation thattakes place as a chain-reaction that results in the formationof peroxides that slowly decompose spontaneously or,much more rapidly, in the presence of catalyzing iron.Therefore, initially, only the most iron-rich lysosomes startto leak and the damage done by released proteolyticenzymes, many of which are partly active (although lessstable) also at neutral pH (Turk et al. 2002 ), is followed byreparative autophagy. If the leak is moderate, the cell maysurvive, while the apoptotic machinery is activated if theleak is more substantial (Antunes et al. 2001 ).

A protective side-effect of ferritin autophagy, in relationto lysosomal and cellular resistance to oxidative stress, isthat ferritin, which is only partly iron-saturated, seems tobe able to temporarily chelate intralysosomal labile ironbefore being degraded itself, thereby reducing its concen-tration of redox-active iron and, therefore, the sensitivity of lysosomes to oxidative stress (Garner et al. 1997 , 1998) .Each molecule of ferritin can store around 4,500 atoms of iron. Consequently, we have postulated that even a limitednumber of autophagocytosed ferritin molecules are able tosubstantially reduce lysosomal redox-active iron (althoughnot the total amount of lysosomal iron). Since autophagyis a continuous process, degraded lysosomal ferritin isconstantly replacedand a state of equilibrium createdbetween cytosolic and lysosomal ferritin. If this is correct,the concentration of lysosomal labile iron is determined by


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the rate of autophagic turnover of ferruginous materials andcytosolic iron-binding proteins, such as ferritin andmetallothioneins, which are a family of iron-binding pro-teins (Baird et al. 2006 ; Garner et al. 1997 , 1998 ; Perssonet al. 2001 ; T. Kurz et al., in preparation). This proposal iscompatible with the fact that cells rich in ferritin, metal-lothioneins and some other stress-induced phase II proteinsare resistant to oxidative stress (Arosio and Levi 2002 ;Baird et al. 2006 ; Garner et al. 1997 ; Viarengo et al. 2000 ).If this hypothesis is correct, there is another side to thecoin. In the case of diseases involving iron-overload, suchas advanced conditions of hemochromatosis, in whichferritin may be iron-saturated, then, rather than acting as abinder of lysosomal redox-active iron, iron-saturated fer-ritin might just increase it and, therefore, enhancelysosomal sensitivity to oxidative stress. These hypothesesare supported by ndings that endocytotic uptake of apo-ferritin, or the autophagy of metallothioneins protectslysosomes against oxidative stress, while the endocytosisof a Fephosphate complex makes them much more vul-nerable to such stress (Fig. 3).

Besides hemochromatosis, an increasing number of diseases have been shown to be associated with the intra-cellular accumulation of iron (in particular withinmitochondria and lysosomes) with consequently enhancedsensitivity to oxidative stress. As pointed out earlier in thisreview, there is a close relation between lysosomes andmitochondria, which is the basis of the lysosomal

mitochondrial axis theories of ageing and apoptosis (Ter-man et al. 2006 ). Because mitochondria are degraded bylysosomes, accumulation of iron inside the former will alsoresult in lysosomal iron loading. Moreover, oxidative stressdue to normal production of hydrogen peroxide by mito-chondria will labilize lysosomes, while released lysosomalenzymes will damage mitochondria and induce furtheroxidative stress.

Friedreichs ataxia is associated with a deciency of frataxin protein, a mitochondrial iron chaperone thatchelates iron in a non-redox-active form and thus protectsfrom Fenton reactions. Frataxin participates in the for-mation of ironsulphur clusters, which are cofactors of several enzymes. Lack of frataxin results in the accumu-lation of mitochondrial redox-active iron with increasedmitochondrial vulnerability to oxidative stress and ensuingneuronal damage (Pandolfo 2006 ). Aceruloplasminemiapatients lack ferroxidase ceruloplasmin activity, whichresults in iron accumulation within a variety of tissues.Neurological symptoms, retinal degeneration, and diabetesfollow (Berg and Youdim 2006 ; Miyajima 2003 ). Side-roblastic anemia is characterized by iron-laden perinuclearmitochondria in developing red blood cells and, some-times, also in neurons and other cell types, due to anenzymatic defect of heme synthesis (Fontenay et al. 2006 ).There is also accumulating evidence that iron accumula-tion within neurons contributes to the development of several common neurodegenerative diseases, such as the

Fig. 5 Lysosomaldestabilization by oxidativestress . The cells antioxidantdefense protects againstmoderate oxidative events.However, if the protectiveshield is overwhelmed,hydrogen peroxide, which ismainly produced bymitochondria, diffuses intolysosomes in abnormalamounts. Since many lysosomesare rich in redox-active iron dueto degradation of iron-containing macromolecules,Fenton-type reactions then takeplace resulting in lysosomalrupture with release of powerfullytic enzymes


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Alzheimers, Parkinsons and Huntingtons diseases (Bergand Youdim 2006 ; Sipe et al. 2002 ).

Leaky lysosomes and apoptosis/necrosis

An active role for lysosomes in the induction of cell deathwas an early suggestion (reviewed in de Duve and Watti-aux 1966 ). Apoptosis or programmed cell death (PCD) wasnot yet identied in those days, although cells with char-acteristics typical for apoptosis had long been described bymorphologists. As pointed out in the introduction to thisreview, the role lysosomes play in apoptosis did notbecome recognized until recentlyand is still being illu-minated. Major reasons for this considerable delay were thefacts that (1) lysosomal membrane permeabilization mayoccur without any apparent ultrastructural alterations, cre-ating an impression, still reected in textbooks, thatlysosomes are sturdy organelles that only break when cellsare dead or almost dead, and that (2) many commonly usedcaspase inhibitors also inhibit cysteine proteases in general,including lysosomal cathepsins (Kroemer and Ja attela2005 ).

The nding a few years ago that some apoptogenicdrugs, which are also lysosomotropic detergents or

aldehydessuch as MSDH ( o-methylserine dodecylamidehydrochloride), sphingosine, Leu-Leu-Ome, and 3-amino-propanalspecically destabilize lysosomes led to afurther focus on the possibility that lysosomal rupture maybe an upstream event in some forms of apoptosis (Boyaet al. 2003 ; Brunk et al. 1997 ; Cirman et al. 2004 ; Kagedalet al. 2001 ). A strong argument for the idea that moderatelysosomal rupture causes apoptosis was the nding that theapoptogenic effects of these lysosomotropic drugs areprevented by an initial exposure to pH-raising lysosomo-tropic substances, for example ammonia or chloroquine(Ohkuma and Poole 1978 ). The raised lysosomal pH pre-vents the intralysosomal accumulation of the apoptogeniclysosomotropic agents (Ka gedal et al. 2001 ; Li et al. 2003 ;Yu et al. 2003a ). The resulting preservation of lysosomalstability and lack of apoptosis certainly links lysosomalleak to apoptosis, although the causal molecular mecha-nisms were not explained by theses experiments.

Oxidative stress during apoptosis, and lysosomalinvolvement in apoptosis due to such stress, is increasinglyrecognized (Brunk et al. 2001 ; Cirman et al. 2004 ; Gui-cciardi et al. 2004 ; Nylandsted et al. 2004 ). The inuenceof such stress on cells depends on the dose.

Figure 6 illustrates the heterogeneity between cells andlysosomes with respect to lysosomal stability following

Fig. 6 Lysosomes are differently sensitive to photooxidation. Usinga 488 nm argon laser, AO-stained J774 cells were depicted byconfocal laser scanning microscopy. Pictures were taken in intervalsof a few minutes during which the samples were subjected to pulsesof blue light . Panel a shows the rst of these ones presenting distinctred uorescence of lysosomes and a weak green uorescence of thenuclei and cytoplasm. In panel b a few cells ( some are arrowed ) showenhanced green uorescence that indicates early lysosomal burst,

while in panel c more advanced lysosomal rupture has resulted in amaximally increased green uorescence. Also note the disappearanceof many lysosomes from the cells. In panels df , the greenuorescence is successively diminishing due to increased plasmamembrane permeability, secondary to the attack of lytic lysosomalenzymes, with escape of cytosolic AO to the surrounding medium.Note some remaining intact lysosomes in a few cells in f


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oxidative stress. It shows J774 cells following photo-oxi-dative stress (AO-loaded cells were exposed to multiplepulses of blue light). Some cells show apoptotic morphol-ogy and the number of intact lysosomes is markedlyreduced, while other cells look almost normal.

Moderate lysosomal rupture induces apoptosis, whilepronounced lysosomal leakage results in necrosis withoutcaspase activation (Zhao et al. 2003 ). The puzzling lack of apoptotic response following major lysosomal damage wasdifcult to explain. It was hypothesized to be a result of aviolent degradation of pro-caspases that, however, couldnot be veried. Very recently, Galaris and coworkerspointed out that a pronounced lysosomal breakup results ina major release to the cytosol of low mass lysosomal ironthat seemed to prevent the activation of caspase-9 withinthe apoptosome that is a complex of pro-caspase-9, cyto-chrome c and ATP. This lack of activation was suggestedto be an effect of iron binding to thiol groups in the activecenter of the pro-caspase (Barbouti et al. 2007 ; Brunk andEaton 2007 ).

As pointed out above, the amount of redox-activelysosomal iron seems to be crucial for lysosomal stabilityduring oxidative stress, probably explaining the variabilityin lysosomal sensitivity to such stress (Nilsson et al. 1997 ).The fact that cells with upregulated metallothioneins, heatshock proteins, or apo-ferritin are relatively resistant tooxidative stress may, at least partially, be explained byautophagic transfer of such macromolecules into the lyso-somal compartment, where they may temporarily bindredox-active iron before being degraded (see above). In theevent that autophagy of such proteins continues, as wouldbe expected as a part of normal turnover, a steady statebetween lysosomal redox-active and non-redox active ironwould be established that is dependent on the cytosolicamounts of these proteins.

Lysosomal enzymes have been shown to induce MMPas well as MPT (mitochondrial permeability transition),through proteolytic activation of phospholipases or the Bcl-2 family members Bid, Bax and Bak. Calpain-like lyso-somal cysteine proteases were found to induce cleavage of Bid (Cirman et al. 2004 ), which in its truncated formrelocates to mitochondria resulting in Bax/Bak activationwith ensuing formation of pores in the outer membrane andresulting escape of cytochrome c. Moreover, cathepsin D,although not cathepsin B, has been reported to modify/ activate Bax to a pore-forming structure (Castino et al.2007 ). Consequently, the release into the cytosol of pro-apoptotic mitochondrial factors such as cytochrome c,apoptosis inducing factor (AIF) and Smac/DIABLO willtrigger cell death (Bidere et al. 2003 ; Brunk et al. 2001 ;Cirman et al. 2004 ; Guicciardi et al. 2004 ; Heinrich et al.2004 ). Moreover, the direct activation of caspases-2 and -8by lysosomal cathepsins has been described for at least

certain cell types (Baumgartner et al. 2007 ; Yeung et al.2006 ), although further studies are needed in order toconrm this.

The mechanisms described above all work through acti-vation of the internal (mitochondrial) pathway of apoptosis.There are, however, also indications that lysosomal desta-bilization is involved in apoptosis following ligation of death receptors on the plasma membrane. Ligation of theFAS receptor was found to give rise to early LMP, althoughneither the mechanisms nor the possible relation to activa-tion of caspase-8 were initially understood (Brunk andSvensson 1999 ). Recently; however, it has been found thatligation of the receptors for tumor necrosis factor andTRAIL, at least in certain types of cells, induces not onlyformation of active caspase-8 from its pro-form, but alsocaspase-8-mediated activation of the pro-apoptotic proteinBax. This pro-apoptotic Bcl-2 family member, which wasformerly believed to be involved only in MMP, was foundalso to participate in LMP, perhaps by forming pores similarto those of the mitochondria (Werneburg et al. 2007 ).

Moreover, the multifunctional cytokine tumor necrosisfactor (TNF) that induces apoptosis has been found toinduce cellular stress, probably by stimulating the pro-duction of superoxide by NADPH oxidase (Autelli et al.2005 ; Frey et al. 2002 ; Kroemer and Ja attela 2005 ; Tanget al. 1999 ). It seems reasonable to believe that theresulting oxidative stress may induce lysosomal iron-mediated rupture with ensuing apoptosis or necrosis.

All these new ndings suggest that lysosomal destabi-lization might play an integral part in apoptosis of both theinternal and external pathways, and suggest that the com-bined action of lysosomal hydrolases and caspases isnecessary for apoptosis. Caspases may be needed for thedegradation of a limited number of specialized proteinsrelevant to apoptosis, such as poly(ADP-ribose)polymeraseand laminin, while lysosomal hydrolases may be neededfor bulk degradation.

A form of apoptosis that is associated with pronouncedautophagy is programmed cell death type II, or autophagiccell death. It is often considered caspase-independent,though whether this is completely correct still remains tobe claried. An alternative interpretation of this phenom-enon, at least in some cases, is that damaged cells try toheal themselves by reparative autophagy, which, in theevent that it fails, is followed by apoptosis. If a majority of cells succeed in their repair efforts, the activation of caspases in only a few cells may be difcult to detect usingbiochemical methods. There are indications of a mutuallyreinforcing link between autophagy and apoptosis,although mutual inhibition under certain conditions hasalso been suggested (Kroemer and Ja attela 2005 ; Maiuriet al. 2007 ). Whether a completely caspase-free type II celldeath does exist still remains to be shown.


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Surprisingly, we recently found (T. Kurz et al., inpreparation) that apoptosis induced by iron depletion fol-lowing exposure to the lipophilic iron-chelator SIH ispreceded by lysosomal destabilization, indicating thatlysosomal damage can be induced by iron overload fol-lowed by oxidative stress as well as by iron depletion. Anyoxidative stress that might occur simultaneously with ironstarvation due to general iron chelation should clearly notinduce lysosomal labilization. Therefore, the destabilizingeffect on lysosomes of iron starvation must be a conse-quence of other apoptogenic stimuli, which are yet to beidentied. The nding nevertheless gives additional sup-port to the emerging view that lysosomal destabilization isa much more common phenomenon in apoptosis than so farrecognized.

Many forms of apoptosis are regulated by activation of the p53 gene. Exactly how the p53 protein triggers theapoptotic cascade is a topic of intense research (Levineet al. 2006 ). If lysosomal destabilization is a general phe-nomenon in apoptosis, it may be assumed that thisphenomenon should be found early in the process of p53activation. A mutated p53 protein exists in a thermo-labileform that is inactive at 37 C but structurally stable andactive at 32 C (Michalovitz et al. 1990 ). When cells thatstably over-express the thermo-labile form of p53 aregrown at 37 C, they remain normal, but when transferredto 32 C they undergo apoptosis within 810 h. Followingtransfer to the permissive temperature of 32 C, these cellsstart to show signs of early lysosomal destabilizationwithin a few hours, long before any apoptotic signs arefound (Yuan et al. 2002 ).

Even if lysosomes seem to be more deeply involved inapoptosis than is generally recognized, many questionsremain to be answered. First among these is whetherlysosomes are regularly destabilized early in forms of apoptosis that are not dependent on oxidative stress, and if so, how. Lysosomal rupture as a result of oxidative stress iswell understood, but why are iron starvation, serum star-vation (Brunk and Svensson 1999 ), and p53 activationfollowed by lysosomal destabilization? The recently dis-covered protein LAPF (for lysosome- associated apoptosis-inducing protein containing PH and F YVE domains), amember of a widespread new family of Phans (proteinscontaining the above domains), was shown to induceapoptosis by attaching itself to lysosomal membranes,causing lysosomal permeabilization and activation of thelysosomalmitochondrial pathway (Chen et al. 2005 ).Perhaps this protein acts as a link between the p53 proteinand lysosomal labilization?

Figure 7 summarizes the role of lysosomes in apoptosisand emphasizes the crosstalk between lysosomes andmitochondria that seems to occur following lysosomalrupture. This crosstalk seems to result in an amplifying

loop with further lysosomal rupture and ensuing mito-chondrial damage.

Lipofuscin and postmitotic ageing

Ageing, a progressive decline in an organisms adaptabilityand consequent increased morbidity and mortality, largelydepends on alterations occurring in long-lived postmitoticcells, such as neurons, cardiac myocytes, and retinal pig-ment epithelial (RPE) cells. These cells are very rarely (ornever) replaced through the division and differentiation of stem cells, thus allowing biological waste materials (suchas lipofuscin, irreversibly damaged mitochondria, andaberrant proteins) to accumulate and gradually substitutenormal structures, leading to functional decay and celldeath (Terman et al. 2007 ). Other ageing effects, some of which are mainly of cosmetic interest, while others aremore serious, include wrinkling of the skin, cataract, andstiffening of blood vessels. These effects are thought to bea consequence of alterations in extra-cellular matrix pro-teins, which undergo polymerization and loose theirelasticity due to glycosylation with accompanying Maillardreactions (binding of carbonyls of reducing sugars toprotein-free amino groups) and ensuing Amadori reorga-nization (the formation of new carbonyls within thecomplex allowing the binding of additional amino groupsand polymerization of proteins to stiff, plastic-like struc-tures) (DeGroot 2004 ). These reactions, resulting in theformation of advanced glycation endproducts (AGEs) havepronounced similarities with formation of lipofuscin insidelysosomes (see below). The difference is that no sugars areinvolved in the case of lipofuscin formation when alde-hydes, formed by degradation of oxidized lipids, act aslinkers between protein residues.

It is now generally accepted that the ageing of long-lived postmitotic cells is induced by endogenously formed(mainly within mitochondria) reactive oxygen species(ROS) causing irreversible damage (mainly to mitochon-dria) with increased oxidative stress and apoptotic celldeath (Barja 2004 ; Harman 1956 ; Ku et al. 1993 ; Lane2005 ; Terman et al. 2006 ). However, if the degradation of damaged structures were to be perfect, no biologicalgarbage would accumulate. Therefore, one has to assumethat damaged structures accumulate with age because theyare not perfectly turned over by the cellular degradationsystems, rst of all lysosomes, which are responsible forthe digestion of a variety of mainly long-lived macromol-ecules and all cellular organelles.

Although it is rapid and effective, lysosomal (autopha-gic) degradation is not completely successful. Even undernormal conditions, some iron-catalyzed peroxidationoccurs intralysosomally (as pointed out, lysosomes are rich


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in redox-active iron), resulting in oxidative modication of autophagocytosed material and yielding lipofuscin (agepigment), a non-degradable, yellowish-brown, autouo-rescent, polymeric compound slowly accumulating withinageing postmitotic cells at a rate that is inversely correlatedto the longevity across species (Brunk and Terman 2002 ).

As pointed out, accumulation of lipofuscin is mainlyseen in long-lived postmitotic cells, but a disease-relatedpigment with similar physico-chemical properties, namedceroid, is also found in cells with a moderate replacementrate as a consequence of reparative autophagy, e.g., in livercells damaged by ionizing radiation or inammation(Ghadially 1975 ). Ceroid also forms due to increasedsensitivity to oxidation, such as in vitamin E deciency(Fattoretti et al. 2002 ) or disturbed lysosomal degradation,e.g., in lysosomal storage diseases (Wenger et al. 2003 ).Due to the variable composition of lysosomal pigment inageing and different diseases, as well as in different tissues,a distinction between lipofuscin and ceroid is reasonableonly from an etiological viewpoint. Since ceroid formsmore quickly than lipofuscin, the latter is perhaps just amore restructured and advanced polymercompare withthe Amadori reorganization of glucosylated proteins.

The fatal lysosomal storage disease, juvenile neuronallipofuscinosis, is caused by a mutation of a lysosomalmembrane protein, which seems to be of importance for thefusion between lysosomes and autophagosomes. As aresult, the inux of degrading enzymes to autophagosomeswill be slow (Cao et al. 2006 ). A delayed degradationallows more time for peroxidation and formation of lipo-fuscin/ceroid. This scenario can be reproduced in cellcultures exposed to inhibitors of lysosomal proteases(delaying degradation) or to moderate oxidative stress(enhancing oxidation) (reviewed in Brunk and Terman2002 ; Terman and Brunk 1998 ).

Lysosomes receive a wide variety of autophagocytosedsubcellular structures, most importantly, mitochondria,which are rich in lipidaceous membrane components andiron-containing proteins, such as cytochrome c. The originof lipofuscin/ceroid from mitochondrial components isproven by the presence of the ATP-synthase subunit c inage pigment or ceroid granules (Elleder et al. 1997 ). Inprofessional scavengers, such as RPE cells and macro-phages (foam cells) in atheroma, a large portion of lipofuscin (or ceroid) originates from endocytosed material(Lee et al. 1998 ; Nilsson et al. 2003 ). Depending on the

Fig. 7 Lysosomalmitochondrial interplay in apoptosis . Activationof the external apoptotic pathway, at least sometimes, results inlysosomal destabilization. This may be a result of direct inuence onlysosomes by caspase 8 or by a coupled production of ceramide that isconverted to the lysosomotropic detergent sphingosine by ceramidase.It may also be an effect of caspase 8-mediated activation of Bax thatattaches itself to, and forms pores in, the lysosomal and outermitochondrial membranes. Hydrogen peroxide, in combination withlysosomal redox-active iron, produces hydroxyl radicals by theFenton reaction leading to peroxidation of the lysosomal membraneand subsequent rupture. The lysosomal membrane can be indirectly

destabilized by the p53 protein in a manner that is still unknown, andby the newly discovered LAPF protein (these events are perhapslinked). Synthetic lysosomal detergents, such as MSDH, andaldehydes, e.g., 3-aminopropanal, labilize lysosomes. Released lyso-somal enzymes can inict further damage on lysosomes directly orvia activation of phospholipases. The internal apoptotic pathway isinitiated by damage to mitochondria (e.g., by activated Bid or Bax,phospholipases or, perhaps, lysosomal enzymes), which results inrelease of cytochrome c and the start of the caspase cascade withensuing apoptosis


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nature of autophagocytosed/endocytosed material, thecomposition of lipofuscin varies amongst different types of postmitotic cells, and no chemical formula can be given forthis complex substance, mainly composed of cross-linkedprotein and lipid residues. It should be pointed out that allforms of lipofuscin contain considerable amounts of redox-active iron, which sensitizes lipofuscin-loaded cells tooxidative stress (Brun and Brunk 1973 ; Brunk and Terman2002 ; Jolly et al. 1995 ).

As already noted, lipofuscin accumulation apparentlyfurther compromises autophagic degradative capacity,prolonging the half-lives of long-lived proteins andorganelles and creating a situation where cells are forced toexercise their functions with less than perfect tools (Fig. 8).Consistent with this idea, the capacity for autophagicdegradation is found to be diminished in aged lipofuscin-loaded cells (Cuervo and Dice 2000 ; Terman 1995 ; Termanet al. 1999b ), which suggests some serious consequences.For example, delayed degradation of mitochondria wouldresult in increased damage by self-produced ROS, addi-tionally contributing to lipofuscinogenesis and, perhaps,inducing apoptotic cell death (see above).

Recently, an elegant study on Caenorhabditis elegansadded new evidences for the hypothesis that lipofuscinaccumulation is causally related to ageing and decline of postmitotic cells. These tiny nematodes are transparent,

which allows a direct measurement of lipofuscin byspectrouorimetry in vivo. It was found that mutantnematodes that lived for longer or shorter than the wildtype, accumulated lipofuscin at a slower or quicker pace,respectively. It was also found that caloric restrictedworms lived longer and accumulated lipofuscin moreslowly than animals fed ad libitum. Finally, when wildtype siblings that aged differently, as evaluated by changesin their motility capacity, were compared, it was foundthat the still mobile and youthful ones at day 11 of theirlife contained only 25% of the lipofuscin that was foundin severely motility-impaired individuals of the sameage. This implies that lipofuscin accumulation reectsbiological rather than chronological age (Gerstbrein et al.2005 ).

Besides the intralysosomal waste material lipofuscin,ageing postmitotic cells accumulate extralysosomal gar-bage, such as damaged dysfunctional mitochondria andindigestible protein aggregates (aggresomes) that for somereason are not autophagocytosed (reviewed in Terman andBrunk 2005b , 2006) . Aged mitochondria are enlarged andshow considerably lowered fusion and ssion activity.Their autophagy may be prevented by their size, sinceautophagy of large structures is apparently energy con-suming, and autophagosomes seem to have an upper limitin volume (Navratil et al. 2008 ; Terman et al. 2007 ).

Fig. 8 The mitochondriallysosomal axis theory of ageing. Thishypothesis tries to explain the relationship between lipofuscinaccumulation, decreased autophagy, increased ROS production, andmitochondrial damage in senescent long-lived postmitotic cells.Mitochondria are damaged by self-produced hydrogen peroxide, atleast if redox-active iron is present. In young cells, damagedmitochondria are rapidly autophagocytosed and degraded becauseenough lysosomal enzymes are distributed throughout the lysosomalcompartment. Small amounts of hydrogen peroxide that normallydiffuse into lysosomes lead to cross-linking of intralysosomal material

and consequent gradual accumulation of lipofuscin in long-livedpostmitotic cells that can neither be degraded nor exocytosed. Bypurely random reasons, senescent cells have an increased amount of lysosomal enzymes directed towards the plentiful lipofuscin-richlysosomes. These enzymes are lost for effective autophagic degra-dation while the lipofusicin remains non-degradable. Theconsequence for the senescent postmitotic cell is further decreasedautophagy, a gradual accumulation of damaged mitochondria,increased hydrogen peroxide production, and apoptotic cell death


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The accumulation of aberrant proteins within ageingpostmitotic cells is a consequence of both ROS-induceddamage and incomplete degradation of altered proteinmolecules. Although damaged proteins may partially pre-serve their functions, their enzymatic activity per unit massdeclines (Rattan 1996 ; Stadtman et al. 1992 ). Aberrantproteins often prove to aggregate. Lewy bodies and neu-robrillary tangles (composed of a-synuclein or thehyperphosphorylated protein tau, respectively) are charac-teristic examples of such aggregates (Bennett 2005 ; Hardyand Selkoe 2002 ).

The importance of autophagy for removal of proteinaggregates and delaying ageing was demonstrated in arecent study on Drosophila (Simonsen et al. 2008 ). Duringnormal ageing of the y, the expression of the Atg8a gene,which is important for autophagy, decreases in neuronsresulting in the accumulation of aggregates of ubiquitinatedprotein. When the expression of Atg8a was upregulated,the aggregates disappeared and the ies showed anincreased resistance to oxidative stress as well as a morethan 50% prolonged lifespan.

There is accumulating evidence that progressive bio-logical garbage accumulation underlies the development of several late-onset diseases, commonly called age-relateddiseases. Age-related macular degeneration is associatedwith a pronounced lipofuscin loading of RPE cells, whichprevents phagocytosis of obsolete photoreceptor outersegments resulting in the degeneration of photoreceptors(Nilsson et al. 2003 ). It is possible that intralysosomalaccumulation of oxidized LDL within iron-rich lysosomesof vessel macrophages may induce foam cell formation andapoptotic cell death due to lysosomal destabilization,contributing to atherosclerosis (Li et al. 2001 , 2006) .Moreover, accumulation of intracellular waste materialsuch as lipofuscin and defective mitochondria within car-diac myocytes results in progressive decline of cardiacfunction leading to heart failure (Terman and Brunk 2005b ;Terman et al. 2008 ).

Formation of indigestible protein aggregates is involvedin a number of age-related neurodegenerative diseases.Alzheimers disease is characterized by the intraneuronalaggregation of protein tau forming neurobrillary tangles(see above) and extraneuronal b-amyloid plaque formation(Hardy and Selkoe 2002 ). There is evidence that b-amyloidplaques may form as a result of neuronal death followingintralysosomal accumulation of amyloid b -protein (Gouraset al. 2005 ; Yu et al. 2005 ; Zheng et al. 2006 ). In Parkin-sons disease dopaminergic neurons of substantia nigraaccumulate a-synuclein aggregates (Yu et al. 2005 ), whileformation of huntingtin aggresomes within striatal neuronsis a characteristic of Huntingtons disease (Lee and Kim2006 ).

Conclusion and perspectives

There is increasing recognition that lysosomes are centralto a great number of cellular functions including not onlythe removal of exhausted organelles but also the metaboliccycling iron and the initiationat least in some casesof apoptotic cell death. Lysosomal dysfunctionsuch asoccurs in ageing and certain lysosomal storage diseasescan be a major factor limiting function and longevity of theorganism. Appreciation of the central role of lysosomes inageing, and certain disease states, may lead to the devel-opment of new therapeutic agents aimed at preserving orcorrecting abnormal lysosomal function.

Acknowledgments We are grateful to Stephen Hampson forexcellent linguistic advice.

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Lysosomes in Iron Metabolism