Lysosomes and Oxidative Stress in Aging and Apoptosis

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Review

Lysosomes and oxidative stress in aging and apoptosis

Tino Kurz a, Alexei Terman b, Bertil Gustafsson c, Ulf T. Brunka,

aDivision of Pharmacology, Faculty of Health Sciences, Linkping University, Linkping, Swedenb Division of Geriatric Medicine, Faculty of Health Sciences, Linkping University, Linkping, Sweden

c Department of Pathology and Cytology, University Hospital, Linkping, Sweden

Received 7 November 2007; received in revised form 13 January 2008; accepted 15 January 2008

Available online 26 January 2008

Abstract

The lysosomal compartment consists of numerous acidic vesicles (pH ~45) 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.

2008 Elsevier B.V. All rights reserved.

Keywords: Aging; Autophagy; Iron; Lipofuscin; Mitochondria; Oxidative stress

1. Introduction

In the late 1950s, Christian de Duve and his coworkers con-

ducted studies of glucose-6-phosphatase while using nonspecific

acid phosphatase as a control enzyme. They found that the latter

enzyme showed latency (enhanced activity with) and had gradient

sedimentation properties close to those of mitochondria. Their

findings strongly suggested that acid phosphatase was localized

within cytosolic membrane-bound vesicles, while glucose-6-

phosphatase was not. With the help of cytochemists and electron

microscopists, particularly Alex Novikoffand his colleagues, it was

later found that a numberof acidichydrolases,capable of degrading

most complex organic molecules, such as proteins, carbohydrates,

lipids and nucleotides, exist together inside an organelle that was

named lysosome (lytic body) [reviewed in [1]]. Lysosomes were

observed to be a heterogeneous group of vacuoles of different size,

form, and density. In the transmission electron microscope, some

lysosomes were seen to contain still recognizable extra- and/orintracellular material under degradation, while others looked ho-

mogeneous or contained electron-dense whorls and clumps.

Soon after its recognition, the lysosomal compartment be-

came thought of as the stomach of the cell, and its importance in

heterophagy (or endocytosis, which includes phagocytosis and

pinocytosis) and autophagy (self-eating) was recognized. Distinct

lysosomes were viewed as storage places for waste products, such

as theagepigment lipofuscin.Such lysosomeswere considered old

and inactive, without remaining lytic capacity, and were named

residual bodies. Later this assumption proved to be wrong, and

such lysosomes are now recognized as integral parts of the

Available online at www.sciencedirect.com

Biochimica et Biophysica Acta 1780 (2008) 12911303www.elsevier.com/locate/bbagen

Abbreviations: ATG, autophagy regulating genes; CMA, chaperone-mediated

autophagy; DFO, desferrioxamine; LDL, low density lipoprotein; LIP, labile iron

pool; LMP, lysosomal membrane permeability; MMP, mitochondrial membrane

permeability; MP, mannose-6-phosphate; MPR, mannose-6-phosphate receptor;

MPT, mitochondrial permeabilitytransition;MSDH, o-methylserine dodecylamide

hydrochloride;ROS, reactive oxygen species; RPE, retinal pigment epithelial; SIH,

salicylaldehyde isonicotinoyl hydrazone; SSM, sulfidesilver method; Tf,

transferrin; TfR, transferrin receptor; TGN, trans-Golgi network Corresponding author. Tel.: +46 13 221515; fax: +46 13 149106.

E-mail address: [email protected] (U.T. Brunk).

0304-4165/$ - see front matter 2008 Elsevier B.V. All rights reserved.doi:10.1016/j.bbagen.2008.01.009
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lysosomal compartment and are known to receive newly produced

lysosomal enzymes in the same way as other lysosomes [2].

Lysosomal enzymes areproduced in thereticular network, matured

in the cis-Golgi apparatus, and transported from the trans-Golgi

network (TGN) within tiny vesicles that fuse with late endosomes

or, perhaps, also with autophagosomes [3]. Mature lysosomes fuse

and divide, allowing their content to be propagated throughout thecompartment [4]. Since lipofuscin, or age pigment, is non-

degradable, the transfer of active hydrolases to lipofuscin-loaded

lysosomes means a waste of enzymes in a futile attempt to degrade

the pigment. In the event that lipofuscin-loaded lysosomesbecome

plentiful, they may deplete autophagolysosomes of sufficient

enzymes and create a lack of degrading capacity. Notwithstanding,

the total amount of lysosomal enzymes may be larger than in

young cells although misdirected.

It was also recognized that many previously known storage

diseases, often with a serious outcome, are due to mutated and

inactive lysosomal hydrolases. As a consequence, substrates that

are non-degradable by the faulty enzymes, accumulate inside thelysosomal compartment. The analogy to lipofuscin accumula-

tion in senescent long-lived postmitotic cells is obvious and,

perhaps, we should look at aging as a storage disease where

lipofuscin is the material that accumulates, although for other

reasons than as a consequence of defective enzymes. Recently it

has been suggested that lysosomal storage diseases, as well as

the aging of long-lived postmitotic cells with resulting lipofuscin

accumulation, disturb normal autophagy and cause an accumu-

lation of polyubiquitinated proteins and dysfunctional mito-

chondria [58]. Consequently, senescence and lysosomal storage

diseases might, possibly, be considered autophagy disorders

(see further below).

Although de Duve made an early suggestion that lysosomesmight be suicide bags [9], they were for a long time considered

sturdy organelles that would not release their potent mixture

of hydrolases until cells were already dead. Lysosomal enzymes

would then be responsible for dissolving dead cells only

(necrosis). This notion was partly based on ultrastructural obser-

vations of seemingly intact lysosomes in devitalized cells. How-

ever, it later turned out that at least the lysosomal enzyme acidic

phosphatase could leave lysosomes that look normal in the

electron microscope [10].

The first reports of lysosomal destabilization or LMP

(lysosomal membrane permeability compare with MMP,

mitochondrial membrane permeability), as an early event inapoptosis came about fifteen years ago. The discovery 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 was then apparently followed by

MMP and classical apoptosis [reviewed in [11]]. Even earlier,

however, it was realized that a mitochondrial release of apopto-

genic substances, such as cytochrome c, activates the caspase

cascade and the internal pathway of apoptosis. Consequently,

almost all researchers within the expanding field of apoptosis

focused on caspases and the mitochondrial mechanisms of ap-

optosis. The vision that lysosomes might play an important role

in at least some forms of apoptosis gathered few advocates

among the more doctrinaire cell death researchers. During the

last two decades, a huge amount of knowledge has accumulated

on apoptosis, but not until it was found that lysosomal calpain-

like cysteine proteases truncate and activate the pro-apoptotic

protein Bid [12] did the role of lysosomes in apoptosis start to

become a somewhat more fashionable field of investigation.

Even though the role of lysosomes in autophagy was re-

cognized early [reviewed in [13]], this phenomenon was longconsidered a murky one. This, however, changed when it was

recently found that autophagy is a much more regulated process

than hitherto believed and under the influence of more than a

score of evolutionarily well-preserved autophagy genes (ATG),

which allow autophagy to be selective and to take three different

forms: macroautophagy, microautophagy, and chaperone-

mediated autophagy (CMA) [reviewed in [14,1517]].

More or less pronounced autophagy commonly occurs in

apoptotic cells. Sometimes such autophagy is extensive, and

apoptosis then deviates from the classical form. This form of

apoptosis has been named autophagic cell death, or apoptosis

type II, and is often considered to be caspase-independent(see further below).

This review will focus on: lysosomal behavior under oxidative

stress; how lysosomes participate in cellular iron metabolism;

how lysosomes and mitochondria interact to induce apoptosis;

and, how lysosomes are involved in postmitotic aging.

2. The function of the lysosomal compartment

The lysosomal compartment has several important functions

and is indispensable for cellular life. Lysosomes thus exist in all

kinds of animal cells except erythrocytes, which have a very

specialized function and a minimal turnover. The degradation of

endocytosed or autophagocytosed materials takes place insidelysosomes that have an acidic (pH ~45) environment, which is

maintained by ATP-dependent proton pumps present in the

lysosomal membrane. Such pumps are also present in the plasma

membrane, especially in tumor cells with their high anaerobic

glycolysis (the Warburg effect) and a resulting need to export

protons to avoid acidification of their own cytosol [18,19].

Following synthesis in the endoplasmic reticulum, lysoso-

mal hydrolases are tagged with mannose-6-phosphate (MP) in

the cis-Golgi area and located into secretory vesicles in the

trans-Golgi area by receptors that ligate MP. The newly

produced hydrolases are transported to slightly acidic late

endosomes, which arise from early endosomes containingendocytosed material, where the enzymes are freed from MP

while the receptors return to the Golgi (see Fig. 1). The late

endosomes then form mature lysosomes, containing material

under degradation, and may fuse with autophagosomes and

other late endosomes to form hybrid organelles. Following

completed degradation of enclosed material, lysosomes turn

into resting ones, which in the electron microscope look

homogeneous and moderately electron-dense. They are then

ready for new rounds of fusion [20].

Following receptor-mediated endocytosis, the initially plas-

ma membrane-bound receptors are often, but not always, re-

circulated back to the plasma membrane, while the ligands are

propagated further down the lysosomal compartment.

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The processing and presentation of antigens in immuno-

competent cells is dependent on a form of endocytotic-exocytotic

activity, while autophagic degradation is of vital importance not

only for the normal turnover of cellular constituents, but also for

the removal of damaged structures and invading microorganisms.

Some cell types are able to exocytose lysosomal contents or even

intact lysosomes (secretory lysosomes) [reviewed in [20]]. It has

been recognized that tumor cells often secrete lysosomal pro-

teases, which, in combination with acidification of their sur-

roundings help them to infiltrateand spread[[2124], reviewed in

[2527]].As pointed out above, lysosomes fuse with autophagosomes

or deliver part of their content (kiss-and-run) to form auto-

phagolysosomes where a variety of organelles and proteins are

degraded into simple components, which in turn are reutilized by

the anabolic machinery of the cell following transport to the

cytosol [reviewed in [28,29]]. Auto- and heterophagosomes may

fuse and create an amphisome, or a hybrid organelle, con-

taining both endogenous and exogenous materials. Moreover,

mature lysosomes fuse and divide, allowing lytic enzymes and

other contents to spread through the lysosomal compartment

(see Fig. 1).

From a physiological point of view, the lysosomal compart-

ment can be looked upon as a box, built from vacuoles that con-

stantly fuse and divide, that receives enzymes from the trans-

Golgi network (TGN) and substrates from either the outside or the

inside of the cell. Most of the mechanisms involved in the

formation of the autophagic double membrane (the phagophore),

inclusion of materials to be degraded, and fusion between auto-

phagosomes and lysosomes was, as mentioned above, recently

worked out as a result of the discovery of a large family of phy-

logenetically well-preserved regulating genes (ATG) [reviewed in

[14,15,17,29]].

As mentioned in the Introduction, autophagy takes place in

three distinct forms: macro-, micro- and chaperone-mediatedautophagy. Of these forms macroautophagy is the best known

and is usually referred to simply as autophagy (for a schematic

description of the lysosomal compartment and related struc-

tures, see Fig. 1).

Following substrate degradation inside individual lyso-

somes, the products are actively transported or diffuse to the

cytosol for reutilization. Autophagy represents a main pathway

for the turnover and reutilization of worn-out long-lived pro-

teins and organelles and is a perfectly normal process. Inter-

estingly, proteasomes, which also play an important role in the

turnover of macromolecules, are themselves degraded by auto-

phagy [30]. The implication of this is that hampered autophagy

might result in defective proteasomes since they, in common

Fig. 1. Autophagycan be subdividedinto three forms: macro-, micro-and chaperone-mediated autophagy(CMA). In macroautophagy, a cup-formed phagophore surrounds

cytosolic organellesthereby creating an autophagosome,which fuseswith a lysosome or a late endosome. Smallportions of cytoplasm enterlysosomesthrough invagination

of the membrane (microautophagy). In CMA, proteins tagged with the amino acid sequence KFERQ are bound by molecular chaperones, e.g. Hsp73, and transported to

lysosomes. The latter form from late endosomes from which they differ in that they do not have mannose-6-phosphate receptors (MPR). Lysosomal hydrolases are

transported bound to MPRs in Golgi-derived vesicles to slightly acidifiedlate endosomesand, possibly, autophagosomes.There, theenzymes arereleasedand theMPRs are

recycled back to the Golgi network.

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with mitochondria and other organelles, are then not properly

renewed.

During periods of starvation, enhanced autophagy serves as a

way for cells to survive by degrading less important parts of

themselves in order to produce building blocks for essential

anabolic events. Subsequent to cellular damage, reparative au-

tophagy follows, in which altered and malfunctioning structuresare replaced. Such reparative autophagy is commonly seen fol-

lowing, for example, ionizing irradiation, virus infection, and

hypoxic or oxidative stress [31,32].

3. The role of lysosomes in cellular iron metabolism and the

stability of lysosomes under oxidative stress

As indicated above, autophagy is the main process that

delivers substrates to the lysosomal compartment in all cells,

except erythrocytes, which lack lysosomes, and professional

scavengers with active heterophagocytosis. Since many macro-

molecules contain iron (e.g., ferritin and mitochondrial electrontransport complexes), the lysosomal compartment is rich in this

essential, although potentially hazardous, transition metal [33

40]. In this respect, lysosomes are unique [reviewed in [41]]. It

is important to recognize that lysosomes that have recently been

engaged in an autophagic degradation of iron-rich compounds

contain high concentrations of iron, while others which have

been inactive for a while may contain only negligible amounts

of it [41,42]. In addition, increased amounts of iron can accu-

mulate in some lysosomes due to accretion of iron-rich non-

degradable materials, such as lipofuscin and, especially, hemo-

siderin. Consequently, the sensitivity of individual lysosomes to

oxidative stress may vary substantially.

Fig. 2 shows the mainly lysosomal distribution of labile ironin HeLa cells using a cytochemical technique, the sulfidesilver

method (SSM). The SSM reveals heavy metals, which normally

means iron since it is the only such metal that normally occurs in

any significant amount in most cells, except in pancreatic -

cells, prostatic epithelial cells and some neurons, all of which

are rich in zinc [37].

Non-dividing cells mainly rely on the turnover and reutli-

zation of iron, while growing populations of cells require its

uptake from their surroundings. The cellular uptake of iron is

tightly controlled and accomplished by a series of sophisticated

events. Iron is carried in the circulation by transferrin (Tf) in a

non-redox-active form. The TfFe complex binds to the trans-

ferrin receptor (TfR), and the TfFeTfR complex is endo-

cytosed and propagated into late endosomes. As a consequence

of the slightly acidic pH of late endosomes, iron is released andtransported to the cytosol by the divalent metal transporter-1

(also known as Nramp 2 or SLC11A2). The TfTfR complex

returns to the plasma membrane, where transferrin is freed from

the TfR and released to the circulation to pick up more iron.

At neutral pH, FeTf binds to the receptor, while Tf does not

(see Fig. 3). In the cytosol, low mass iron binds to certain

specific proteins, which in turn up-regulate ferritin and down-

regulate TfR, by interfering with the translation of their

mRNA. By these mechanisms, cytosolic iron is kept at the

lowest possible level that allows synthesis of necessary iron-

containing macromolecules [43].

Even though it is not known exactly how low mass iron istransported from lysosomes to the cytosol following autophagic

degradation of iron-containing macromolecules, it may be

assumed that mechanisms akin to those that transport iron from

late endosomes are involved. It is also not known whether iron

that is bound to divalent metal transporters is redox-active.

Recently, an alternative mechanism for the delivery of iron

from late endosomes/lysosomes to mitochondria was suggested.

It involves a direct transport between these two types of orga-

nelles following temporary close contact (kiss and run) [44].

For a schematic view of the involvement of lysosomes in iron

metabolism, see Fig. 3.

Rapidly growing tumor cells have a high demand for iron and,

therefore, iron chelators may be used to stop their proliferationand induce apoptosis. Encouraging therapeutic results have been

obtained in phase III studies using specific iron chelators, which

seem to have a selective effect on tumor cells, while not harming

normal cells that also need iron, e.g., erythroblasts [45].

Some investigators have found supportfor the hypothesis that

when low mass iron is required, it is released directly from

ferritin within the cytosol [4648]. Others favour an alternative

mechanism for iron release, namely autophagy followed by

Fig. 2. Cytochemical demonstration of lysosomal iron in HeLa cells using the high pH, high S2 sulfidesilver method (SSM). After 25 min of development (A) only a

few dark-stained iron-containing lysosomes are visible (arrows), indicating particularly iron-rich lysosomes, whereas after 40 min of development (B) a strong andwidespread lysosomal-type pattern is found, indicating at least some iron in most lysosomes.



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transport of released iron to the cytosol [[36,3840,49,50] and

Kurz et al. in preparation]. The latter hypothesis is supported by

findings of many electron microscopists who over the years have

noticed ferritin inside autophagosomes [51]. Another argumentfor the autophagy hypothesis is that exposure of cells in culture

to a panel of inhibitors of lysosomal enzymes prevents the

release of iron from ferritin [50]. Moreover, exposure of cells to

the iron chelator DFO, which is endocytosed and transported

into the lysosomal compartment where it remains undegraded

and acts as a sink for iron, rapidly results in a major decrease of

cytosolic labile (calcein-chelatable) iron [49]. The latter finding

supposedly reflects a rapid incorporation of cytosolic iron into

ferritin or iron-containing macromolecules under construction.

Such a rapid disappearance of calcein-chelatable iron from the

cytosol is in agreement with the notion that cytosolic labile iron

is in rapid transit and kept at the lowest possible concentration inorder to avoid Fenton-type reactions [43]. If iron were liberated

from ferritin directly in the cytosol, one would expect a stable

cytosolic concentration of chelatable iron for quite a while. How-

ever, whether autophagy or a mechanism that allows the release

of iron directly from ferritin act in concert, or if one of them is

dominant, remains to be established.

As mentioned above, excessive iron may accumulate in-

tralysosomally as a component of lipofuscin [[52], and reviewed

in [53]] or in the form of hemosiderin. The latter substance seems

to represent a particularly iron-rich form of lipofuscin composed

of non-degradable polymerized aldehyde-bridged ferritin resi-

dues. Because cells do not have the capacity to rid themselves

of such intralysosomal iron, it slowly accumulates over time, even

if the uptake is effectively regulated. This accumulation is par-

ticularly evident in the reticulo-endothelial system, in hepato-

cytes, and in long-lived postmitotic cells, such as neurons and

cardiac myocytes. It seems probable that the sensitivity to oxi-dative stress is enhanced in such cells.

As long as Fe(II) has at least one of its six coordinates free, it

is capable of catalyzing a homolytic splitting of hydrogen

peroxide resulting in the formation of the extremely reactive

hydroxyl radical (Fenton reaction).

Fe2 H2O2Fe3 HOd OH

The favourable conditions for intralysomal Fenton reactions

are a function of their low internal pH (~45) and the presence

of reducing equivalents, such as cysteine [5456]. At this pH,

cysteine easily reduces iron(III) to iron(II). Another reducingagent inside autophagolysosomes may be the superoxide anion

radical (O2U). Mitochondria undergoing autophagic degrada-

tion probably continue to produce these radicals for a while,

perhaps even in increased amounts, following disabling of the

mitochondrial electron transport complexes secondary to the

proteolytic attack.

Hydrogenperoxideforms continuouslyin cells, mainly by loss

of electrons from the mitochondrial electron transport chain, but

also from a variety of oxido-reductases that operate by one-

electron transfers. Some hydrogen peroxide may escape the

effective cytosolic degradation by catalase and glutathione per-

oxidase and diffuse into lysosomes [57], which do not contain

these enzymes (those that are autophagocytosed are rapidly

Fig. 3. Cellular iron metabolism. In serum, iron is bound in a transferriniron complex that attaches to transferrin receptors, which are endocytosed. In late endosomes,

at a reduced pH, iron is released 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 affinity chelators before it is either used in the synthesis of iron-containing proteins or stored in ferritin. The LIP also

receives iron from lysosomes following degradation of iron-containing proteins, e.g. ferritin and mitochondrial electron transport complexes.



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degraded), to react with labile iron and form hydroxyl radicals,

or similarly reactive ferryl or perferryl compounds [reviewed in

[58]] (see Fig. 4).

In normal conditions, with a low and steady state rate of auto-

phagy, the result of the inevitable Fenton-type reactions inside

lysosomes is the slow formation of lipofuscin (age pigment) over

time. Following enhanced autophagy, for example because ofreparative efforts, when often oxidative stress is present as well, a

much more rapid formation of the pigment occurs. In such cases

that are unrelated to normal aging, the pigment is called ceroid,

although both lipofuscin and ceroid basically have the same

composition and mode of origin [53,59].

If enhanced oxidative stress results in peroxidation of the

lysosomal membrane, lysosomes become leaky after a delay

that is dependent on the magnitude of oxidative stress. This

happens also if cells are exposed only briefly to oxidative stress

before being returned to standard culture conditions without

further stress. Consequently, any satisfactory hypothesis on the

mechanisms behind oxidative stress-induced lysosomal rupturemust provide firm and distinct links between the triggering

events, which occur within minutes, and the ultimate leak,

which may be delayed by hours. This link seems to be lysoso-

mal membrane peroxidation that takes place as a chain reaction

following its start. This reaction results in the formation of

peroxides that slowly decompose spontaneously or, more ra-

pidly, in the presence of catalyzing iron. Initially, only the most

iron-rich lysosomes start to leak and the damage done by re-

leased proteolytic enzymes, many of which are partly active

also at neutral pH [60] is followed by reparative autophagy. If

the leak is moderate the cell may survive, while the apoptotic

machinery is activated if the leak is more substantial [61].

A protective side-effect of ferritin autophagy, in relation to

lysosomal and cellular resistance to oxidative stress, is that

ferritin which is only partly iron-saturated seems to be able

to temporarily chelate intralysosomal labile iron before being

degraded, thereby reducing its concentration and, therefore,

the sensitivity of lysosomes to oxidative stress [62,63]. Each

molecule of ferritin can store around 4500 atoms of iron. Con-sequently, a limited number of autophagocytosed ferritin mo-

lecules would be able to substantially reduce lysosomal redox-

active iron (although not the total amount of lysosomal iron).

Since autophagy is a continuous process, degraded lysosomal

ferritin would constantly be replaced and a state of equi

librium created between cytosolic and lysosomal ferritin. If so,

hypothetically, the concentration of lysosomal labile iron would

be determined by the rate of autophagic turnover of ferruginous

materials and cytosolic iron-binding proteins, such as ferritin

and metallothionein, which is another iron-binding protein

[[56,6264] and Kurz et al. in preparation]. This proposal is

compatible with the fact that cells rich in ferritin, metallothio-neins and some other stress-induced phase II proteins are re-

sistant to oxidative stress [56,62,65,66]. If this hypothesis is

correct, there is, however, another side of the coin. In the case of

diseases involving iron overload, such as advanced conditions

of hemochromatosis, in which ferritin is more or less iron-

saturated, then, rather than acting as a protector of lysosomes,

iron-rich ferritin might increase lysosomal redox-active iron and,

therefore, enhance lysosomal sensitivity to oxidative stress.

Besides hemochromatosis, an increasing number of diseases

have been shown to depend on an intracellular accumulation

of iron (in particular within mitochondria and lysosomes)

with consequently enhanced sensitivity to oxidative stress. As

Fig. 4. Lysosomal destabilization by oxidative stress.The cell'santioxidant defense protects against most oxidative events. However, if the protectiveshield is overwhelmed,

hydrogen peroxide, which is mainly produced by mitochondria, diffuses into lysosomes in abnormal amounts. Since many lysosomes are rich in redox-active iron due todegradation of iron-containing macromolecules, Fenton-type reactions then take place resulting in lysosomal rupture with release of powerful lytic enzymes.



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pointed out earlier in this review, there is a close relation be-

tween lysosomes and mitochondria, which is the basis of our

theories relating the lysosomalmitochondrial axis to aging and

apoptosis [67]. Because the latter ones are degraded by the

former, accumulation of iron inside mitochondria will also re-

sult in lysosomal iron-loading. Moreover, oxidative stress due

to normal production of hydrogen peroxide by mitochondriawill labilize lysosomes, while released lysosomal enzymes will

damage mitochondria and induce further oxidative stress.

Friedreich's ataxia is associated with a deficiency of the

frataxin protein, a mitochondrial iron chaperone that chelates

iron in a non-redox-active form and thus protects from Fenton

reactions. Frataxin participates in the formation of ironsulfur

clusters, which are cofactors of several enzymes. Lack of fra-

taxin results in the accumulation of mitochondrial redox-active

iron with increased mitochondrial vulnerability to oxidative

stress and ensuing neuronal damage [68]. Aceruloplasminemia

patients lack ferroxidase ceruloplasmin activity, which results

in iron accumulation within a variety of tissues. Neurologicalsymptoms, retinal degeneration, and diabetes follow [69,70].

Sideroblastic anemia is characterized by iron-laden perinu-

clear mitochondria in developing red blood cells and, some-

times, also in neurons and other cell types, due to an enzymatic

defect of heme synthesis [71]. There is also accumulating evi-

dence that intraneuronal iron accumulation contributes to the

development of several common neurodegenerative diseases,

such as the Alzheimer's, Parkinson's and Huntington's diseases

[69,72].

4. Lysosomes and apoptosis

A role for lysosomes in the active induction of cell death washypothesized by Christian de Duve soon after his discovery of

lysosomes, which he nicknamed suicide bags [reviewed in

[73]]. In those days, apoptosis or programmed cell death (PCD)

was not yet identified, although cells with typical apoptotic

characteristics had long been described by morphologists. As

pointed out in the introduction to this review, the role lysosomes

play in apoptosis did not become recognized until recently.

Major reasons for this delay were the facts (i) that lysosomal

membrane permeabilization may occur without any apparent

ultrastructural alterations, creating an impression, still reflected

in some textbooks, that lysosomes are sturdy organelles that

only break when cells are already dead or almost dead, and (ii)that many commonly used caspase inhibitors also inhibit other

cysteine proteases, including lysosomal cathepsins [74].

The finding that some apoptogenic drugs which are also lysoso-

motropic detergents or aldehydes suchas MSDH(o-methylserine

dodecylamide hydrochloride), sphingosine, Leu-Leu-OMe or

3-aminopropanal specifically destabilize lysosomes led to a

further focus on the possibility that lysosomal rupture may be

an upstream event in some forms of apoptosis [12,7577]. The

apoptogenic effects of these drugs are prevented by an initial

exposure to pH-raising lysosomotropic substances, for exam-

ple ammonia and chloroquine [78], which would inhibit the

intralysosomal accumulation of the apoptogenic lysosomotro-

pic agents [76,79,80].

Oxidative stress during apoptosis, and lysosomal involve-

ment in apoptosis due to such stress, is being increasingly re-

cognized [11,12,81,82]. Moderate lysosomal rupture induces

apoptosis, while pronounced lysosomal leak results in necrosis

without caspase activation [83]. The lack of apoptotic response

following major lysosomal damage may be a result of the di-

gestion of pro-caspases or may be secondary to a pronouncedleakage of lysosomal iron that prevents activation of caspase-9

within the apoptosome [84].

As pointed out above, the amount of redox-active lysosomal

iron seems to be crucial for lysosomal stability during oxidative

stress, probably explaining the variability in lysosomal sensiti-

vity to such stress [42]. The fact that cells with up-regulated

metallothioneins, heat shock proteins, or apo-ferritin are re-

latively resistant to oxidative stress might be at least partially

explained by autophagic transfer of these macromolecules

into the lysosomal compartment where they may temporarily

bind iron in a non-redox-active form before being degraded

(see above). In the event that autophagy of such proteins con-tinues, a new steady state between redox-active and non-redox

active lysosomal iron would be established.

Lysosomal enzymes have been shown to induce MPT (mito-

chondrial permeability transition) either directly or indirectly,

through proteolytic activation of phospholipases or the Bcl-2

family members Bid, Bax and Bak. Calpain-like lysosomal

cysteine proteases induce cleavage of Bid [12], which in its

truncated form relocates to mitochondria resulting in Bax/Bak

activation, while cathepsin D has been reported to activate both

Bid and Bax. Consequently, pro-apoptotic mitochondrial factors

such as cytochrome c, apoptosis-inducing factor (AIF) and

Smac/DIABLO release into the cytosol will each trigger cell

death [11,12,81,85,86]. Moreover, activation of caspases-2 and-8 by lysosomal cathepsins has been described for at least

certain cell types [87,88].

The mechanisms described above, all work through activa-

tion of the internal (mitochondrial) pathway of apoptosis. There

are, however, also indications that lysosomal destabilization is

involved in apoptosis following ligation of death receptors on

the plasma membrane. Ligation of the FAS receptor was found

to give rise to early LMP, although neither the mechanisms nor

the possible relation to activation of caspase-8 were understood

[89]. Recently, however, it was shown that ligation of the re-

ceptors for tumor necrosis factor and TRAIL, at least in certain

types of cells, induces not only formation of active caspase-8from its pro-form, but also caspase-8-mediated activation of the

pro-apoptotic protein Bax that belongs to the Bcl-2 family. Bax,

in turn, formerly believed to be involved only in MMP, was

found also to participate in LMP, probably by forming pores

similar to those of the mitochondria [90]. These findings

suggest that lysosomal destabilization might be an integral part

in apoptosis of both the internal and external pathways, and that

the combined action of lysosomal hydrolases and caspases is

necessary for apoptosis.

A form of apoptosis that is associated with pronounced auto-

phagy is named programmed cell death type II, or autopha-

gic cell death, and is often considered caspase-independent. It

should be pointed out, however, that activation of autophagy



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does not necessarily mean realization of PCD-II. Being an im-

portant cell repair process, which is activated in response to

different stressors such as oxidants, radiation, starvation, infec-

tion, etc., autophagy protects cells from death if the damage to

cellular structures is not too advanced and still can be healed. If

cellular damage is irreversible, autophagy can act as a PCD

mechanism, providing for massive degradation of damagedcellular constituents [91]. Whether a completely caspase-free

type II cell death does exist remains to be shown. There are

indications of a mutually reinforcing link between autophagy

and apoptosis, although mutual inhibition under certain condi-

tions has also been suggested [92].

Surprisingly, it was recently found (unpublished results) that

apoptosis induced by iron depletion is preceded by lysosomal

destabilization, indicating that lysosomal damage can be in-

duced both by iron overload followed by oxidative stress and by

iron depletion. Any oxidative stress that possibly might occur

simultaneously with iron starvation should not induce lysoso-

mal labilization in the presence of firmly chelated redox-activeiron. Therefore, the destabilizing effect on lysosomes of iron

starvation must be a consequence of other apoptogenic stimuli.

The identity of these presently unknown stimuli is yet to be dis-

covered, but the finding gives additional support to the emerg-

ing view that lysosomal destabilization is a much more common

phenomenon in apoptosis than so far recognized.

Many forms of apoptosis are regulated by activation of the

p53 gene. Exactly how the p53 protein triggers the apoptotic

cascade is a topic of intense research [93]. If lysosomal labili-

zation is a general phenomenon in apoptosis it may be assumed

that such labilization should be found early in the process of p53

activation. An engineered p53 protein exists in a thermo-labile

form that is inactive at 37 C but structurally stable and active at32C [94]. When cells that stably over-express the thermo-labile

form of p53 are grown at 37 C, they remain normal, but when

transferred to 32 C they undergo apoptosis within 810 h.

Following transfer to the permissive temperature, these cells

start showing signs of early lysosomal destabilization within a

few hours, long before any apoptotic signs are found [95].

Even if lysosomes seem to be more deeply involved in

apoptosis than is generally recognized, many questions remain to

be answered. First among those is whether lysosomes are re-

gularly destabilized early in apoptosis that is not dependent on

oxidative stress, and if so, how? Lysosomal rupture as a result of

oxidative stress is well understood, but why are iron starvation,serum starvation [89] or p53 activation followed by lysosomal

destabilization? The recently discovered protein LAPF (for ly-

sosome-associated apoptosis-inducing protein containing PH

and FYVE domains), a member of a widespread new family of

Phafins (proteins containing the above domains), was shown to

induce apoptosis by attaching itself to lysosomal membranes,

Fig. 5. Lysosomalmitochondrial interplay in apoptosis. At least in several forms of apoptosis, lysosomes and mitochondria interact. Activation of the external

apoptotic pathway may result in lysosomal destabilization, either directly by caspase 8 or indirectly by a coupled production of ceramide that is converted to

sphingosine, or by a caspase 8-mediated truncation and activation of Bax that attaches itself to, and forms pores in, the lysosomal and outer mitochondrial membranes.

Sphingosine is a lysosomotropic detergent. Hydrogen peroxide, in combination with lysosomal redox-active iron, produces hydroxyl radicals by the Fenton reaction

leading to peroxidation of the lysosomal membrane and subsequent rupture. The lysosomal membrane can be indirectly destabilized by p53 in a manner that is still

unknown, and by a newly discovered protein (LAPF). Synthetic lysosomal detergents, such as MSDH, and aldehydes, e.g. 3-aminopropanal, labilize lysosomes.

Released lysosomal enzymes can inflict further damage to lysosomes directly or via activation of phospholipases. The internal apoptotic pathway is initiated by

damage to mitochondria (e.g. by activated Bid or Bax, phospholipases or lysosomal enzymes), which results in release of cytochrome c and the start of the caspasecascade with ensuing apoptosis.



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causing lysosomal permeabilization and activation of the lyso-

somalmitochondrial pathway [96]. Perhaps this protein acts as a

link between the p53 protein and lysosomal labilization?

Fig. 5 summarizes the role of lysosomes in apoptosis and

emphasizes the crosstalk between lysosomes and mitochondria

that seems to occur following lysosomal rupture, resulting in an

amplifying loop with further lysosomal rupture and ensuingmitochondrial damage.

5. Lysosomes and postmitotic aging

Aging, a progressive decline in an organism's adaptability

and consequent increased morbidity and mortality, largely de-

pends on alterations occurring in long-lived postmitotic cells,

such as neurons, cardiac myocytes, and retinal pigment epithe-

lial (RPE) cells. These cells are very rarely (or never) replaced

due to division and differentiation of stem cells, allowing accu-

mulation of biological waste materials (such as lipofuscin, irre-

versibly damaged mitochondria, and aberrant proteins) thatgradually replace normal structures, leading to functional decay

and cell death [28].

It is now generally accepted that aging of long-lived postmi-

totic cells is induced by endogenously formed (mainly from

mitochondria) reactive oxygen species (ROS) causing irrever-

sible damage (mainly to mitochondria) with increased oxidative

stress and apoptotic cell death [67,97100]. However, if degra-

dation of damaged structures were to be perfect, no biological

garbage would accumulate. Therefore, one has to assume that

damaged structures accumulate with age because they are not

perfectly turned over by the cellular degradation systems, first of

all lysosomes, which are responsible for digestion of a variety of

mainly long-lived macromolecules and all cellular organelles.

Although rapid and effective, lysosomal (autophagic) de-

gradation is not completely successful. Even under normal

conditions, some iron-catalyzed peroxidation occurs intralyso-

somally (as pointed out, lysosomes are rich in redox-active iron),resulting in oxidative modification of autophagocytosed mate-

rial and yielding lipofuscin (age pigment), a non-degradable,

yellowish-brown, autofluorescent, polymeric compound slowly

accumulating within aging postmitotic cells at a rate that is

inversely correlated to the normal life span of the organism [53].

As pointed out, accumulation of lipofuscin is mainly seen in

long-lived postmitotic cells, but a disease-related pigment with

similar physico-chemical properties, named ceroid, is also found

in cells with a moderate replacement rate as a consequence of

reparative autophagy, e.g., in liver cells damaged by ionizing

radiation or inflammation [101]. Ceroid also forms due to in-

creased oxidative stress, such as in vitamin E deficiency [102] ordisturbed lysosomal degradation, e.g., in lysosomal storage di-

seases [103]. Due to the variable composition of lysosomal

pigment in aging and different diseases, as well as in different

tissues, the distinction between lipofuscin and ceroid is reason-

able only from an etiological viewpoint. Since ceroid forms

more quickly than lipofuscin, the former maybe is a less mature

form of the latter.

The fatal lysosomal storage disease, juvenile neuronal

lipofuscinosis, is caused by a mutation of a lysosomal mem-

brane protein, which seems to be of importance for the fusion

Fig. 6. The mitochondriallysosomal axis theoryof agingdescribes the relationshipbetween lipofuscin accumulation, decreased autophagy, increased ROS production, and

mitochondrial damage in senescent long-lived postmitotic cells. Mitochondria are damaged by self-produced hydrogen peroxide. In young cells, damaged mitochondria are

rapidly autophagocytosed and degraded because enough lysosomal enzymes are distributed throughout the lysosomal compartment. Small amounts of hydrogen peroxide

that normally diffuse into lysosomes lead to cross-linking of intralysosomal material and consequent gradual accumulation of lipofuscin in long-lived postmitotic cells that

neither canbe degraded norexocytosed. Senescentcells direct an increasedamount of lysosomalenzymes towards lipofuscin-rich lysosomes in a fruitless attemptto degrade

this material. These enzymes, however, are lost for effective autophagic degradation. The consequence for the senescent postmitotic cell is further decrease of autophagicdegradation, a gradual accumulation of damaged mitochondria, increase in hydrogen peroxide production, oxidative stress, and apoptotic cell death.



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between lysosomes and autophagosomes. As a result, the influx

of degrading enzymes to autophagosomes will be slow [104]. A

delayed degradation allows more time for peroxidation and

formation of lipofuscin/ceroid. This scenario can be reproduced

in cell cultures exposed to inhibitors of lysosomal proteases or

to moderate oxidative stress [reviewed in [53,59]].

Lysosomes receive a wide variety of autophagocytosed sub-cellular structures, most importantly, mitochondria, which are

rich in lipidaceous membrane components and iron-containing

proteins, such as cytochrome c. The assumption that lipofuscin/

ceroid forms from mitochondrial components is supported by

the presence of the ATP-synthase subunit c in age pigment or

ceroid granules [105]. In professional scavengers, such as RPE

cells and macrophages (foam cells) in atheroma, a large portion

of lipofuscin (or ceroid) originates from endocytosed material

[106,107]. Depending on the nature of autophagocytosed/endo-

cytosed material, the composition of lipofuscin varies amongst

different types of postmitotic cells, and no chemical formula can

be given for this complex substance, mainly composed of cross-linked protein and lipid residues. It should be pointed out that

lipofuscin contains considerable amounts of redox-active iron,

which sensitizes lipofuscin-loaded cells to oxidative stress

[52,53,108].

As remarked before, lipofuscin accumulation apparently

further compromises autophagic degradative capacity, prolong-

ing the half-lives of long-lived proteins and organelles and cre-

ating a situation where cells are forced to exercise their functions

with less than perfect tools. Consistent with this idea, the

capacity for autophagic degradation is found to be diminished in

aged lipofuscin-loaded cells [7,109,110], which suggests some

serious consequences. For example, delayed degradation of

mitochondria would result in increased damage by self-pro-duced ROS, additionally contributing to lipofuscinogenesis and,

perhaps, inducing apoptotic cell death (see above and Fig. 6).

Besides the intralysosomal waste material lipofuscin, aging

postmitotic cells accumulate extralysosomal garbage, such

as damaged dysfunctional mitochondria and indigestible pro-

tein aggregates (aggresomes) that for some reason are not auto-

phagocytosed [reviewed in [111,112]]. Aged mitochondria are

enlarged and show considerably lowered fusion and fission

activity. Their autophagy may be prevented by their size, since

autophagy of large structures is apparently energy consuming,

and autophagosomes seem to have an upper limit in volume

[28,113].The accumulation of aberrant proteins within aging post-

mitotic cells [114,115] is a consequence of both ROS-induced

damage and incomplete degradation of altered protein mole-

cules. Although damaged proteins may partially preserve their

functions, their enzymatic activity per unit mass declines

[114,115]. Lewy bodies and neurofibrillary tangles (composed

of-synuclein or the hyperphosphorylated protein tau, respec-

tively) are characteristic examples of such aggregates [116,117].

There is accumulating evidence that progressive biological

garbage accumulation underlies the development of several

late-onset diseases, commonly called age-related diseases. Age-

related macular degeneration is associated with a pronounced

lipofuscin loading of RPE cells, which prevents phagocytosis of

obsolete photoreceptor outer segments resulting in the degen-

eration of photoreceptors [107]. It is possible that intralysoso-

mal accumulation of oxidized LDL within iron-rich lysosomes

of vessel macrophages may induce foam cell formation and

apoptotic cell death due to lysosomal destabilization, contribut-

ing to atherosclerosis [118,119]. Excessive accumulation of

intracellular waste material such as lipofuscin and defectivemitochondria within cardiac myocytes results in progressive

decline of cardiac function leading to heart failure [111].

Formation of indigestible protein aggregates is involved in a

number of age-related neurodegenerative diseases. Alzheimer's

disease is characterized by the intraneuronal aggregation of pro-

tein tau forming neurofibrillary tangles (see above) and extra-

neuronal -amyloid plaque formation [116]. There is evidence

that-amyloid plaques may form as a result of neuronal death

following intralysosomal accumulation of amyloid -protein

[120122]. In Parkinson's disease dopaminergic neurons of sub-

stantia nigra accumulate -synuclein aggregates [117], while

formation of huntingtin aggresomes within striatal neurons is acharacteristic of Huntington's disease [123].

Acknowledgement

We are grateful to Stephen Hampson for excellent linguistic

advice.

References

[1] C. de Duve, The lysosome turns fifty, Nat. Cell Biol. 7 (2005) 847849.

[2] U. Brunk, J.L. Ericsson, Electron microscopical studies on rat brain

neurons. Localization of acid phosphatase and mode of formation of

lipofuscin bodies, J. Ultrastruct. Res. 38 (1972) 115.

[3] W.A. Dunn Jr., Studies on the mechanisms of autophagy: maturation of

the autophagic vacuole, J. Cell Biol. 110 (1990) 19351945.

[4] U. Brunk, Distribution and shifts of ingested marker particles in residual

bodies and other lysosomes. Studies on in vitro cultivated human glia

cells in phase II and 3, Exp. Cell Res. 79 (1973) 1527.

[5] K. Kiselyov, J.J. Jennigs Jr., Y. Rbaibi, C.T. Chu, Autophagy,

mitochondria and cell death in lysosomal storage diseases, Autophagy

3 (2007) 259262.

[6] C. Settembre, A. Fraldi, L. Jahreiss, C. Spampanato, C. Venturi, D.

Medina, R.D. Pablo, C. Tacchetti, D.C. Rubinsztein, A. Ballabio, A block

of autophagy in lysosomal storage disorders, Hum. Mol. Genet. 17 (2008)

119129.

[7] A. Terman, The effect of age on formation and elimination of autophagic

vacuoles in mouse hepatocytes, Gerontology 41 (1995) 319326.

[8] A. Terman, U.T. Brunk, The aging myocardium: roles of mitochondrial

damage and lysosomal degradation,Heart LungCirc. 14 (2005) 107114.

[9] C. de Duve, Lysosomes, a new group of cytoplasmic particles, in: T.

Hayashi (Ed.), Subcellular Particles, The Ronald Press Co., New York,

1959, pp. 128159.

[10] U.T. Brunk, J.L. Ericsson, Cytochemical evidence for the leakage of acid

phosphatase through ultrastructurally intact lysosomal membranes,

Histochem. J. 4 (1972) 479491.

[11] U.T. Brunk, J. Neuzil, J.W. Eaton, Lysosomal involvement in apoptosis,

Redox Rep. 6 (2001) 9197.

[12] T. Cirman, K. Oresic, G.D. Mazovec, V. Turk, J.C. Reed, R.M. Myers, G.S.

Salvesen, B. Turk, Selective disruption of lysosomes in HeLa cells triggers

apoptosis mediated by cleavage of Bid by multiple papain-like lysosomal

cathepsins, J. Biol. Chem. 279 (2004) 35783587.

[13] H. Glaumann, J.L. Ericsson, L. Marzella, Mechanisms of intralysosomal

degradation with special reference to autophagocytosis and heteropha-

gocytosis of cell organelles, Int. Rev. Cytol. 73 (1981) 149182.



11/13

[14] T. Shintani, D.J. Klionsky, Autophagy in health and disease: a double-

edged sword, Science 306 (2004) 990995.

[15] T. Yorimitsu, D.J. Klionsky, Autophagy: molecular machinery for self-

eating, Cell Death Differ. 12 (2005) 15421552.

[16] A.M. Cuervo, E. Bergamini, U.T. Brunk, W. Droge, M. Ffrench, A.

Terman, Autophagy and aging: the importance of maintaining clean

cells, Autophagy 1 (2005) 131140.

[17] K. Suzuki, Y. Ohsumi, Molecular machinery of autophagosome for-mation in yeast, Saccharomyces cerevisiae, FEBS Lett. 581 (2007)

21562161.

[18] G.L. Semenza, D. Artemov, A. Bedi, Z. Bhujwalla, K. Chiles, D. Feldser,

E. Laughner, R. Ravi, J. Simons, P. Taghavi, H. Zhong, The metabolism

of tumours: 70 years later, Novartis Found. Symp. 240 (2001) 251260.

[19] R. Bartrons, J. Caro, Hypoxia, glucose metabolism and the Warburg's

effect, J. Bioenerg. Biomembr. 39 (2007) 223229.

[20] J.P. Luzio, P.R. Pryor, N.A. Bright, Lysosomes: fusion and function, Nat.

Rev., Mol. Cell Biol. 8 (2007) 622632.

[21] R. Martinez-Zaguilan,R.M. Lynch,G.M. Martinez,R.J. Gillies, Vacuolar-

type H(+)-ATPases are functionally expressed in plasma membranes of

human tumor cells, Am. J. Physiol. 265 (1993) C1015C1029.

[22] P. Montcourrier, I. Silver, R. Farnoud, I. Bird, H. Rochefort, Breast cancer

cells have a high capacity to acidify extracellular milieu by a dual

mechanism, Clin. Exp. Metastasis 15 (1997) 382392.[23] A. De Milito, S. Fais, Tumor acidity, chemoresistance and proton pump

inhibitors, Future Oncol. 1 (2005) 779786.

[24] K. Lorenzo, P. Ton,J.L. Clark,S. Coulibaly, L. Mach, Invasive propertiesof

murinesquamouscarcinoma cells: secretion of matrix-degrading cathepsins

is attributable to a deficiency in the mannose 6-phosphate/insulin-like

growth factor II receptor, Cancer Res. 60 (2000) 40704076.

[25] H. Rochefort, F. Capony, M. Garcia, Cathepsin D: a protease involved in

breast cancer metastasis, Cancer Metastasis Rev. 9 (1990) 321331.

[26] I. Podgorski, B.F. Sloane, Cathepsin B and its role(s) in cancer progres-

sion, Biochem. Soc. Symp. (2003) 263276.

[27] B.C. Victor, B.F. Sloane, Cysteine cathepsin non-inhibitory binding

partners: modulating intracellular trafficking and function, Biol. Chem.

388 (2007) 11311140.

[28] A. Terman, B. Gustafsson, U.T. Brunk, Autophagy, organelles and

ageing, J. Pathol. 211 (2007) 134143.[29] D.J. Klionsky, Autophagy: from phenomenology to molecular under-

standing in less than a decade, Nat. Rev., Mol. Cell Biol. 8 (2007)

931937.

[30] A.M. Cuervo,A. Palmer, A.J. Rivett, E. Knecht, Degradation of proteasomes

by lysosomes in rat liver, Eur. J. Biochem. 227 (1995) 792800.

[31] S. Kaushik, A.M. Cuervo, Autophagy as a cell-repair mechanism:

activation of chaperone-mediated autophagy during oxidative stress, Mol.

Aspects Med. 27 (2006) 444454.

[32] E. Bergamini, Autophagy: a cell repair mechanism that retards ageing and

age-associated diseases and can be intensified pharmacologically, Mol.

Aspects Med. 27 (2006) 403410.

[33] H. Miyawaki, Histochemistry and electron microscopy of iron-containing

granules, lysosomes, and lipofuscin in mouse mammary glands, J. Natl.

Cancer Inst. 34 (1965) 601623.

[34] A. Brun, U. Brunk, Histochemical indications for lysosomal localizationof heavy metals in normal rat brain and liver, J. Histochem. Cytochem. 18

(1970) 820827.

[35] I. Schraufsttter, P.A. Hyslop, J.H. Jackson, C.G. Cochrane, Oxidant-

induced DNA damage of target cells, J. Clin. Invest. 82 (1988) 10401050.

[36] I. Sakaida, M.E. Kyle, J.L. Farber, Autophagic degradation of protein

generates a pool of ferric iron required for the killing of cultured

hepatocytes by an oxidative stress, Mol. Pharmacol. 37 (1990) 435442.

[37] J.M. Zdolsek, K. Roberg, U.T. Brunk, Visualization of iron in cultured

macrophages: a cytochemical light and electron microscopic study using

autometallography, Free Radic. Biol. Med. 15 (1993) 111.

[38] D.C. Radisky, J. Kaplan, Iron in cytosolic ferritin can be recycled through

lysosomal degradation in human fibroblasts, Biochem. J. 336 (1998)

201205.

[39] S. Roberts, A. Bomford, Ferritin iron kinetics and protein turnover in

K562 cells, J. Biol. Chem. 263 (1988) 1918119187.

[40] B. Vaisman, E. Fibach, A.M. Konijn, Utilization of intracellular ferritin

iron for hemoglobin synthesis in developing human erythroid precursors,

Blood 90 (1997) 831838.

[41] T. Kurz, A. Terman, U.T. Brunk, Autophagy, ageing and apoptosis: the

role of oxidative stress and lysosomal iron, Arch. Biochem. Biophys. 462

(2007) 220230.

[42] E. Nilsson, R. Ghassemifar, U.T. Brunk, Lysosomal heterogeneity be-

tween and within cells with respect to resistance against oxidative stress,Histochem. J. 29 (1997) 857865.

[43] M. Kruszewski, Labile iron pool: the main determinant of cellular re-

sponse to oxidative stress, Mutat. Res. 531 (2003) 8192.

[44] A.S. Zhang, A.D. Sheftel, P. Ponka, Intracellular kinetics of iron in

reticulocytes: evidence for endosome involvement in iron targeting to

mitochondria, Blood 105 (2005) 368375.

[45] D.R. Richardson, Therapeutic potential of iron chelators in cancer

therapy, Adv. Exp. Med. Biol. 509 (2002) 231249.

[46] H. Takagi, D. Shi, Y. Ha, N.M. Allewell, E.C. Theil, Localized unfolding

at the junction of three ferritin subunits. A mechanism for iron release?

J. Biol. Chem. 273 (1998) 1868518688.

[47] X. Liu, W. Jin, E.C. Theil, Opening protein pores with chaotropes

enhances Fe reduction and chelation of Fe from the ferritin biomineral,

Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 36533658.

[48] I. De Domenico, M.B. Vaughn, L. Li, D. Bagley, G. Musci, D.M. Ward, J.Kaplan, Ferroportin-mediated mobilization of ferritin iron precedes

ferritin degradation by the proteasome, EMBO J. 25 (2006) 53965404.

[49] M. Tenopoulou, P.T. Doulias, A. Barbouti, U. Brunk, D. Galaris, Role of

compartmentalized redox-active iron in hydrogen peroxide-induced DNA

damage and apoptosis, Biochem. J. 387 (2005) 703710.

[50] T.Z. Kidane, E. Sauble, M.C. Linder, Release of iron from ferritin requires

lysosomal activity, Am. J. Physiol., Cell Physiol. 291 (2006) 445455.

[51] A.M. Koorts,M. Viljoen, Ferritin andferritin isoforms I: structurefunction

relationships, synthesis, degradationand secretion, Arch. Physiol. Biochem.

113 (2007) 3054.

[52] R.D. Jolly, B.V. Douglas, P.M. Davey, J.E. Roiri, Lipofuscin in bovine

muscle and brain: a model for studying age pigment, Gerontology 41

(1995) 283295.

[53] U.T. Brunk, A. Terman, Lipofuscin: mechanisms of age-related accumulation

and influence on cell function, Free Radic. Biol. Med. 33 (2002) 611619.[54] R.L. Pisoni, T.L. Acker, K.M. Lisowski, R.M. Lemons, J.G. Thoene, A

cysteine-specific lysosomal transport system provides a major route for

the delivery of thiol to human fibroblast lysosomes: possible role in sup-

porting lysosomal proteolysis, J. Cell Biol. 110 (1990) 327335.

[55] F.Q. Schafer, G.R. Buettner, Acidic pH amplifies iron-mediated lipid

peroxidation in cells, Free Radic. Biol. Med. 28 (2000) 11751181.

[56] S.K. Baird, T. Kurz, U.T. Brunk, Metallothionein protects against oxidative

stress-induced lysosomal destabilization, Biochem. J. 394 (2006) 275283.

[57] J.H. Walton, H.A. Lewis, The partition coefficients of hydrogen peroxide

between water and certain organic solvents. J. Am. Chem. Soc. 38 (1916)

633638.

[58] J.W. Eaton, M. Qian, Molecular bases of cellular iron toxicity, Free Radic.

Biol. Med. 32 (2002) 833840.

[59] A. Terman, U.T. Brunk, Lipofuscin: mechanisms of formation and in-

crease with age, APMIS 106 (1998) 265276.[60] B. Turk, V. Stoka, J. Rozman-Pungercar, T. Cirman, G. Droga-Mazovec, K.

Oresic, V. Turk, Apoptotic pathways: involvement of lysosomal proteases,

Biol. Chem. 383 (2002) 10351044.

[61] F. Antunes, E. Cadenas, U.T. Brunk, Apoptosis induced by exposure to a

low steady-state concentration of H2O2 is a consequence of lysosomal

rupture, Biochem. J. 356 (2001) 549555.

[62] B. Garner, W. Li, K. Roberg, U.T. Brunk, On the cytoprotective role of

ferritin in macrophages and its ability to enhance lysosomal stability, Free

Radic. Res. 27 (1997) 487500.

[63] B. Garner, K. Roberg, U.T. Brunk, Endogenous ferritin protects cells with

iron-laden lysosomes against oxidative stress, Free Radic. Res. 29 (1998)

103114.

[64] H.L. Persson, K.J. Nilsson, U.T. Brunk, Novel cellular defenses against

iron and oxidation: ferritin and autophagocytosis preserve lysosomal sta-

bility in airway epithelium, Redox Rep. 6 (2001) 5763.



12/13

[65] A. Viarengo, B. Burlando, N. Ceratto, I. Panfoli, Antioxidant role of metal-

lothioneins: a comparative overview, Cell. Mol. Biol. 46 (2000) 407417.

[66] P. Arosio, S. Levi, Ferritin, iron homeostasis, and oxidative damage, Free

Radic. Biol. Med. 33 (2002) 457463.

[67] A. Terman, B. Gustafsson, U.T. Brunk, The lysosomalmitochondrial

axis theory of postmitotic aging and cell death, Chem. Biol. Interact. 163

(2006) 2937.

[68] M. Pandolfo, Iron and Friedreich ataxia, J. Neural Transm., Suppl. (2006)143146.

[69] D. Berg, M.B. Youdim, Role of iron in neurodegenerative disorders, Top.

Magn. Reson. Imaging 17 (2006) 517.

[70] H. Miyajima, Aceruloplasminemia, an iron metabolic disorder, Neuro-

pathology 23 (2003) 345350.

[71] M. Fontenay, S. Cathelin, M. Amiot, E. Gyan, E. Solary, Mitochondria in

hematopoiesisand hematologicaldiseases, Oncogene25 (2006) 47574767.

[72] J.C. Sipe, P. Lee, E. Beutler, Brain iron metabolism and neurodegenera-

tive disorders, Dev. Neurosci. 24 (2002) 188196.

[73] C. de Duve, R. Wattiaux, Functions of lysosomes, Annu. Rev. Physiol. 28

(1966) 435492.

[74] G. Kroemer, M. Jttel, Lysosomes and autophagy in cell death control,

Nat. Rev. Cancer 5 (2005) 886897.

[75] U.T. Brunk, H. Dalen, K. Roberg, H.B. Hellquist, Photo-oxidative

disruption of lysosomal membranes causes apoptosis of cultured humanfibroblasts, Free Radic. Biol. Med. 23 (1997) 616626.

[76] K. Kgedal, M. Zhao, I. Svensson, U.T. Brunk, Sphingosine-induced

apoptosis is dependent on lysosomal proteases, Biochem. J. 359 (2001)

335343.

[77] P. Boya, K. Andreau, D. Poncet, N. Zamzami, J.L. Perfettini, D. Metivier,

D.M. Ojcius, M. Jttel, G. Kroemer, Lysosomal membrane permeabi-

lization induces cell death in a mitochondrion-dependent fashion, J. Exp.

Med. 197 (2003) 13231334.

[78] S. Ohkuma, B. Poole, Fluorescence probe measurement of the

intralysosomal pH in living cells and the perturbation of pH by various

agents, Proc. Natl. Acad. Sci. U. S. A. 75 (1978) 33273331.

[79] W. Li, X.M. Yuan, S. Ivanova, K.J. Tracey, J.W. Eaton, U.T. Brunk,

3-Aminopropanal, formed during cerebral ischaemia, is a potent lysoso-

motropic neurotoxin, Biochem. J. 371 (2003) 429436.

[80] Z. Yu, W. Li, U.T. Brunk, 3-Aminopropanal is a lysosomotropic aldehydethat causes oxidativestress and apoptosis by rupturing lysosomes, APMIS

111 (2003) 643652.

[81] M.E. Guicciardi, M. Leist, G.J. Gores, Lysosomes in cell death,

Oncogene 23 (2004) 28812890.

[82] J. Nylandsted, M. Gyrd-Hansen, A. Danielewicz, N. Fehrenbacher, U.

Lademann, M. Hoyer-Hansen, E. Weber, G. Multhoff, M. Rohde, M.

Jttel, Heat shock protein 70 promotes cell survival by inhibiting lyso-

somal membrane permeabilization, J. Exp. Med. 200 (2004) 425435.

[83] M. Zhao, F. Antunes, J.W. Eaton,U.T.Brunk, Lysosomal enzymes promote

mitochondrial oxidant production, cytochrome c release and apoptosis, Eur.

J. Biochem. 270 (2003) 37783786.

[84] A. Barbouti, C. Amorgianiotis, E. Kolettas, P. Kanavaros, D. Galaris,

Hydrogen peroxide inhibits caspase-dependent apoptosis by inactivating

procaspase-9 in an iron-dependent manner, Free Radic. Biol. Med. 43

(2007) 13771387.[85] N. Bidere, H.K. Lorenzo, S. Carmona, M. Laforge, F. Harper, C. Dumont,

A. Senik, Cathepsin D triggers Bax activation, resulting in selective

apoptosis-inducing factor (AIF) relocation in T lymphocytes entering the

early commitment phase to apoptosis, J. Biol. Chem. 278 (2003)

3140131411.

[86] M. Heinrich, J. Neumeyer, M. Jakob, C. Hallas, V. Tchikov, S. Winoto-

Morbach, M. Wickel, W. Schneider-Brachert, A. Trauzold, A. Hethke, S.

Schutze, Cathepsin D links TNF-induced acid sphingomyelinase to Bid-

mediated caspase-9 and -3 activation,Cell DeathDiffer. 11(2004) 550563.

[87] B.H. Yeung, D.C. Huang, F.A. Sinicrope, PS-341 (bortezomib) induces

lysosomal cathepsin B release and a caspase-2-dependent mitochondrial

permeabilization and apoptosis in human pancreatic cancer cells, J . Biol.

Chem. 281 (2006) 1192311932.

[88] H.K. Baumgartner, J.V. Gerasimenko, C. Thorne, L.H. Ashurst, S.L.

Barrow, M.A. Chvanov, S. Gillies, D.N. Criddle, A.V. Tepikin, O.H.

Petersen, R. Sutton, A.J. Watson, O.V. Gerasimenko, Caspase-8-mediated

apoptosis induced by oxidativestressis independentof theintrinsic pathway

and dependent on cathepsins, Am. J. Physiol.: Gastrointest. Liver Physiol.

293 (2007) G296G307.

[89] U.T. Brunk, I. Svensson, Oxidative stress, growth factor starvation and

Fas activation may all cause apoptosis through lysosomal leak, Redox

Rep. 4 (1999) 311.

[90] N.W. Werneburg, M.E. Guicciardi, S.F. Bronk, S.H. Kaufmann, G.J.Gores, Tumor necrosis factor-related apoptosis-inducing ligand activates

a lysosomal pathway of apoptosis that is regulated by Bcl-2 proteins,

J. Biol. Chem. 282 (2007) 2896028970.

[91] W. Bursch, The autophagosomal-lysosomal compartment in programmed

cell death, Cell Death Differ. 8 (2001) 569581.

[92] M.C. Maiuri, E. Zalckvar, A. Kimchi, G. Kroemer, Self-eating and self-

killing: crosstalk between autophagy and apoptosis, Nat. Rev., Mol. Cell

Biol. 8 (2007) 741752.

[93] A.J. Levine, W. Hu, Z. Feng, The P53 pathway: what questions remain to

be explored? Cell Death Differ. 13 (2006) 10271036.

[94] D. Michalovitz, O. Halevy, M. Oren, Conditional inhibition of trans-

formation and of cell proliferation by a temperature-sensitive mutant of

p53, Cell 62 (1990) 671680.

[95] X.M. Yuan, W. Li, H. Dalen, J. Lotem, R. Kama, L. Sachs, U.T. Brunk,

Lysosomal destabilization in p53-induced apoptosis, Proc. Natl. Acad.Sci. U. S. A. 99 (2002) 62866291.

[96] W. Chen,N. Li,T. Chen, Y. Han, C. Li,Y. Wang, W. He,L. Zhang, T. Wan,

X. Cao, The lysosome-associated apoptosis-inducing protein containing

the pleckstrin homology (PH) and FYVEdomains (LAPF), representative

of a novel family of PH and FYVE domain-containing proteins, induces

caspase-independent apoptosisvia the lysosomalmitochondrial pathway,

J. Biol. Chem. 280 (2005) 4098540995.

[97] D. Harman, Aging: a theory based on free radical and radiation chemistry,

J. Gerontol. 211 (1956) 298300.

[98] G. Barja, Free radicals and aging, Trends Neurosci. 27 (2004) 595600.

[99] N. Lane, Power, Sex, Suicide: Mitochondria and the Meaning of Life,

Oxford University Press, Oxford, 2005, pp. 289301.

[100] H.H. Ku, U.T. Brunk, R.S. Sohal, Relationship between mitochondrial

superoxide and hydrogen peroxide production and longevity of mamma-

lian species, Free Radic. Biol. Med. 15 (1993) 621627.[101] F.N. Ghadially, Ultrastructural Pathology of the Cell and Matrix, Second

ed., Butterworths, London, 1975.

[102] P. Fattoretti, C. Bertoni-Freddari, T. Casoli, G. Di Stefano, M. Solazzi, E.

Corvi, Morphometry of age pigment (lipofuscin) and of ceroid pigment

deposits associated with vitamin E deficiency, Arch. Gerontol. Geriatr. 34

(2002) 263268.

[103] D.A. Wenger, S. Coppola, S.L. Liu, Insights intothe diagnosisand treatment

of lysosomal storage diseases, Arch. Neurol. 60 (2003) 322328.

[104] Y. Cao, J.A. Espinola, E. Fossale, A.C. Massey, A.M. Cuervo, M.E.

MacDonald, S.L. Cotman, Autophagy is disrupted in a knock-in mouse

modelof juvenile neuronal ceroid lipofuscinosis, J. Biol. Chem. 281 (2006)

2048320493.

[105] M. Elleder, J. Sokolova, M. Hrebicek, Follow-up study of subunit c

of mitochondrial ATP synthase (SCMAS) in Batten disease and in

unrelated lysosomal disorders, Acta Neuropathol. (Berl) 93 (1997)379390.

[106] F.Y. Lee, T.S. Lee, C.C. Pan, A.L. Huang, L.Y. Chau, Colocalization of

iron and ceroid in human atherosclerotic lesions, Atherosclerosis 138

(1998) 281288.

[107] S.E. Nilsson, S.P. Sundelin, U. Wihlmark, U.T. Brunk, Aging of cultured

retinal pigment epithelial cells: oxidative reactions, lipofuscin formation

and blue light damage, Doc. Ophthalmol. 106 (2003) 1316.

[108] A. Brun, U. Brunk, Heavy metal localization and age related accu-

mulation in the rat nervous system. A histochemical and atomic absorp-

tion spectrophotometric study, Histochemie 34 (1973) 333342.

[109] A. Terman, H. Dalen, U.T. Brunk, Ceroid/lipofuscin-loaded human

fibroblasts show decreased survival time and diminished autophagocy-

tosis during amino acid starvation, Exp. Gerontol. 34 (1999) 943957.

[110] A.M. Cuervo, J.F. Dice, When lysosomes get old, Exp. Gerontol. 35

(2000) 119131.



13/13

[111] A. Terman, U.T. Brunk, Autophagy in cardiac myocyte homeostasis,

aging, and pathology, Cardiovasc. Res. 68 (2005) 355365.

[112] A. Terman, U.T. Brunk, Oxidative stress, accumulation of biological

garbage, and aging, Antioxid. Redox Signal. 8 (2006) 197204.

[113] M. Navratil, A. Terman, E.A. Arriaga, Giant mitochondria do not fuse

and exchange their contents with normal mitochondria, Exp. Cell Res.

314 (2008) 164172.

[114] E.R. Stadtman, P.E. Starke-Reed, C.N. Oliver, J.M. Carney, R.A. Floyd,Protein modification in aging, EXS 62 (1992) 6472.

[115] S.I. Rattan, Synthesis, modifications, and turnover of proteins during

aging, Exp. Gerontol. 31 (1996) 3347.

[116] J. Hardy, D.J. Selkoe, Theamyloid hypothesisof Alzheimer'sdisease: progress

and problems on the road to therapeutics, Science 297 (2002) 353356.

[117] M.C. Bennett, The role of alpha-synuclein in neurodegenerative diseases,

Pharmacol. Ther. 105 (2005) 311331.

[118] W. Li, H. Dalen, J.W. Eaton, X.M. Yuan, Apoptoticdeath of inflammatory

cells in human atheroma, Arterioscler. Thromb. Vasc. Biol. 21 (2001)

11241130.

[119] W. Li, M. Ostblom, L.H. Xu, A. Hellsten, P. Leanderson, B. Liedberg,

U.T. Brunk, J.W. Eaton, X.M. Yuan, Cytocidal effects of atheromatous

plaque components: the death zone revisited, FASEB J. 20 (2006)

22812290.

[120] W.H. Yu, A.M. Cuervo, A. Kumar, C.M. Peterhoff, S.D. Schmidt, J.H.

Lee,P.S.Mohan, M. Mercken,M.R. Farmery, L.O. Tjernberg, Y. Jiang, K.

Duff, Y. Uchiyama, J. Naslund, P.M. Mathews, A.M. Cataldo, R.A.

Nixon, Macroautophagya novel beta-amyloid peptide-generating path-way activated in Alzheimer's disease, J. Cell Biol. 171 (2005) 8798.

[121] L. Zheng, K. Roberg,F. Jerhammar, J. Marcusson, A. Terman, Autophagy

of amyloid beta-protein in differentiated neuroblastoma cells exposed to

oxidative stress, Neurosci. Lett. 394 (2006) 184189.

[122] G.K. Gouras, C.G. Almeida, R.H. Takahashi, Intraneuronal Abeta

accumulation and origin of plaques in Alzheimer's disease, Neurobiol.

Aging 26 (2005) 12351244.

[123] S.T. Lee, M. Kim, Aging and neurodegeneration. Molecular mechanisms

of neuronal loss in Huntington's disease, Mech. Ageing Dev. 127 (2006)

432435.


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Lysosomes and Oxidative Stress in Aging and Apoptosis