COMPREHENSIVE INVITED REVIEWS
Mitochondrial Turnover and Aging
of Long-Lived Postmitotic Cells:
The Mitochondrial–Lysosomal Axis Theory of Aging
Alexei Terman,1Tino Kurz,2Marian Navratil,3Edgar A. Arriaga,3and Ulf T. Brunk2
It is now generally accepted that aging and eventual death of multicellular organisms is to a large extent related
to macromolecular damage by mitochondrially produced reactive oxygen species, mostly affecting long-lived
postmitotic cells, such as neurons and cardiac myocytes. These cells are rarely or not at all replaced during life
and can be as old as the whole organism. The inherent inability of autophagy and other cellular-degradation
mechanisms to remove damaged structures completely results in the progressive accumulation of garbage,
including cytosolic protein aggregates, defective mitochondria, and lipofuscin, an intralysosomal indigestible
material. In this review, we stress the importance of crosstalk between mitochondria and lysosomes in aging.
The slow accumulation of lipofuscin within lysosomes seems to depress autophagy, resulting in reduced
turnover of effective mitochondria. The latter not only are functionally deficient but also produce increased
amounts of reactive oxygen species, prompting lipofuscinogenesis. Moreover, defective and enlarged mito-
chondria are poorly autophagocytosed and constitute a growing population of badly functioning organelles that
do not fuse and exchange their contents with normal mitochondria. The progress of these changes seems to
result in enhanced oxidative stress, decreased ATP production, and collapse of the cellular catabolic machinery,
which eventually is incompatible with survival. Antioxid. Redox Signal. 12, 503–535.
II. ROS, Mitochondrial Damage, and Aging
A. Biomolecular damage under normal conditions
B. Imperfect turnover of damaged biologic structures
C. Major targets of ROS attack: mitochondria and lysosomes
III. Mitochondrial Fusion, Fission, and Biogenesis
A. The role of mitochondrial dynamics
B. Mitochondrial fusion
C. Mitochondrial fission
D. Mitochondrial biogenesis
IV. Mitochondrial Proteolytic Systems
V. Mitochondrial Turnover by Autophagy
A. The main functions of the lysosomal compartment
C. Autophagic degradation of mitochondria (mitophagy)
VI. Lipofuscin Formation and Its Influence on Autophagy
A. Influence of labile iron and ROS on lipofuscin formation
B. Consequences of the nondegradability of lipofuscin
C. Disease-related accumulation of intralysosomal and extralysosomal waste
Reviewing Editors: Enrique Cadenas, Sandra Cardoso, Joanne Clark, Mark Hannink, Sergio Papa, George Perry,
Rodrigue Rossignol, and Raj S. Sohal
1Department of Clinical Pathology and Cytology, Karolinska University Hospital, Huddinge, Stockholm, Sweden.
2Department of Pharmacology, Faculty of Health Sciences, Linko ¨ping University, Linko ¨ping, Sweden.
3Department of Chemistry, University of Minnesota, Minneapolis, Minnesota.
ANTIOXIDANTS & REDOX SIGNALING
Volume 12, Number 4, 2010
ª Mary Ann Liebert, Inc.
VII. Imperfect Mitochondrial Turnover and Postmitotic Cellular Aging
A. Age-related accumulation of defective mitochondria within postmitotic cells
B. Age-related decline in autophagy and Lon protease activity accelerates mitochondrial damage
C. Enlarged mitochondria are resistant to degradation and do not fuse with normal ones
D. Mechanisms of the age-related accumulation of mitochondria with homoplasmic mtDNA mutations
E. Decreased mitochondrial biogenesis in aged cells
VIII. Summary and Conclusions
and the fountain of eternal youth sought after, ever since the
beginnings of human reflection on life and death. During the
short documented period of human history that is available to
us, numerous theories on biologic aging, or senescence (and
how it may be prevented) have been advanced, debated, and,
in most cases, rejected (156, 187, 251, 255). Now, however,
some agreement seems to exist that cellular oxidation and
oxygen-derived radicals contribute to biologic aging (hereaf-
terreferred toasaging), whichcanbedefined asaprogressive
decline in an organism’s adaptability, followed by a conse-
quent increase in morbidity and mortality (48, 221). The
oxidative-stress theory of aging, although still far from pro-
ven, is presently one of the major aging hypotheses, even
though its details are vaguely outlined, the conclusions are
often obscure, and attempts to prevent aging by antioxidants
are so far unsuccessful (10, 98, 213).
The amalgamation for metabolic symbiosis of anaero-
bic methane-producing bacteria and bacterial ancestors of
present-day mitochondria into a prototype chimeric eukary-
otic cell resulted in a capacity for much-enhanced energy
most successful unification of two different forms of bacteria,
this amalgamation created organisms with substantially bet-
ter access to energy than their ancestors. The transformation,
however, had the inevitable side effect of exposing early eu-
karyotic cells to reactive oxygen species (ROS). These species,
which have electrons that escape by accident from the mito-
chondrial electron-transporting system as their main cause of
origin, may, in the presence of redox-active transition metals,
damage a large variety of macromolecules by transforming
them into dysfunctional and non-degradable garbage that
results in cellular functional decay and, eventually, in cell
All cells are not alike in this respect, however. Most pro-
nounced age-related changes occur in long-lived postmitotic
cells, such as neurons, retinal pigment epithelium (RPE),
cardiac myocytes, and skeletal muscle fibers. These cells are
all highly vulnerable to aging due, of course, to their intensive
oxygen metabolism and a consequent extensive ROS pro-
duction; this is especially true for cardiac myocytes, cortical
neurons, and RPE cells (91). A no-less-important contribution
to vulnerability of long-lived postmitotic cells to aging is the
fact that these cells are replaced rarely, or not at all, and can
thus be as old as the organism itself (19). In contrast, short-
lived postmitotic cells, which are frequently replaced because
of division and differentiation of stem cells (e.g., intestinal
epithelial cells and peripheral blood cells), do not accumulate
s can be seen from the 5,000-year-old Sumerian Gilga-
mesh epos, the reasons for aging have been pondered,
substantial amounts of waste during their short lifetimes.
However, such short-lived postmitotic cells may alter to some
extent with organismal age, possibly reflecting changes in
stem and progenitor cells, even though their continuous di-
vision considerably decreases their intracellular accumulation
of waste products.
Recently it was shown that the proliferation potential of
stem and progenitor cells decreases with age (218, 219). Be-
cause of this deterioration, the efficiency of biologic waste
dilution by cell division also decreases in stem and progenitor
cells with age, accompanied by the less-frequent replacement
of mature short-lived postmitotic cells. It follows that stem
cells, previously believed to escape aging, acquire over time
some of the properties of aged long-lived postmitotic cells, in
particular increased lipofuscin-related autofluorescence, ele-
vated carbonyl content, and enhanced oxidative stress (218,
219). Conceivably, stem and progenitor cells, along with
mature short-lived postmitotic cells, may then have to rely on
a defective lysosomal compartment, the function of which is
hampered by the presence of lipofuscin (see Section VI.B),
which affects the turnover of essential structures and macro-
It should be added that stem and progenitor cells are also
prone to the accumulation of mutations that are reproduced
during cell division and that may result in the development of
neoplasms. The majority of tumors thus arise in actively
proliferating cell populations that are characterized by rela-
tively high numbers of stem and progenitor cells. Tumor bi-
ology is, however, a separate age-related problem and not a
subject of this review. A comparison between short-lived and
long-lived postmitotic cells is given in Table 1.
Taking into consideration the plethora of symptoms that
appears at advanced age, including hormonal and immu-
nologic dysfunction, how could a decline in the function and
the eventual death of postmitotic cells explain a major por-
tion of those symptoms? Part of the answer to this question
may lie in the comparatively small number of commanding
neuroendocrine cells in the hypothalamus. By their pro-
duction of tropic hormones, these postmitotic cells regulate
the outflow of a number of secondary-order hormones from
the pituitary gland, which in turn regulate a range of tertiary-
order hormones from peripheral endocrine glands at the
bottom of the pyramid. It is conceivable that the age-related
loss of a limited number of commanders at the top of this
pyramid could lead to an overthrow of the whole organism.
However, further discussion of the hormonal and immune
systems and their relation to aging is not within the scope of
Aging may thus be assumed to be, to a large extent, a result
of the deterioration of long-lived postmitotic cells due to their
limited renewal capacity, even if oxidative damage to the
components of connective tissues, which normally are re-
504 TERMAN ET AL.
cycled by matrix metalloproteinases (86), also contributes to
the aging process. The modification of connective tissue
components, making them non-degradable, results from
metal-dependent oxidation, or from glycation with secondary
Amadori rearrangements into advanced glycation end prod-
ucts (AGEs). In either case, degeneration of the cartilages and
ligaments, decreased elasticity of the skin and arteries, and
hardening of the lens with resulting presbyopia occur. Fur-
thermore, age-related defects in the remodeling of connective
tissues contributes to declines in bone structural integrity,
back problems, and the development of arthritic joints (248,
250). Even if these age-related problems are not life threat-
ening, they can be sources of much frustration for the elderly,
take alook inthe mirror,findtheirblood pressure elevated, or
try to read a book without glasses. Hence, the recent finding
that the elasticity, at least partly, may be restored by agents
that break cross-links, including those of AGEs (268), may be
of great clinical significance.
How does oxidation damagepostmitotic cells?Basically, as
long as oxidative injuries can be properly repaired, no axi-
omatic need exists for such damage to occur. All cells, post-
mitotic ones included, are fantastic self-repairing machines
that turn over and reuse the building blocks of their macro-
molecular constituents. However, the occurrence of age-
related damage implies that the cellular renewal mechanisms
are not perfect (i.e., not all damaged structures are being re-
moved and, as a result, they gradually accumulate in the cell).
The decline in vigor seems significantly to accelerate at old
age, suggesting that the defective turnover and repair of
damaged structures, preventing successful rejuvenation,
progresses with age.
It is known that aging is characterized by the increasing
accumulation in long-lived postmitotic cells of dysfunctional,
usually enlarged (sometimes called giant) mitochondria,
lipofuscin-loaded lysosomes, and oxidatively modified cyto-
solic proteins and lipids. Damaged proteins often accumulate
215).Sincethe emergence ofthe oxidativestress orfree radical
theory of aging, such alterations have been considered the
result of a gradual accumulation of oxidatively injured mac-
romolecules. Some other theories, such as the somatic muta-
tion theory of aging (33, 54) and the error catastrophe theory
(175), emphasized instead the role of the erroneous synthesis
of macromolecules in aging. Later studies, however, did not
show any substantial increase in the occurrence of synthetic
errors with age (83, 97). Although somatic mutations do ac-
cumulate, they cannot explain the variety of changes associ-
ated with aging (120). Apparently, the role of somatic
mutations is mostly restricted to the increased frequency of
malignant neoplasms with age (see earlier).
Because damaged structures obviously would not accu-
mulate if they were being perfectly removed, it can be rea-
soned that it is not the formation of dysfunctional and
oxidized proteins and lipids that creates all the multifaceted
problems that exist for aged long-lived postmitotic cells, but
rather the malfunction of catabolic enzymes, such as the cy-
tosolic proteasomes and calpains and the host of lysosomal
enzymes that cannot completely degrade damaged struc-
tures. With this line of reasoning, aging, together with a
number of neurodegenerative diseases, is starting to be con-
sidered a catabolic disorder.
Having provideda general description ofthe current under-
standing of aging, this review now focuses on mitochondrial
Table 1. Renewal and Age-related Changes of Terminally Differentiated (Postmitotic) Cells
with Different Life Spans
Characteristic Short-lived postmitotic cellsLong-lived postmitotic cells
Mature enterocytes, peripheral blood cells Neurons, cardiac myocytes, skeletal muscle
fibers, RPE cells
Long, often comparable with
that of the whole organism
Similar to that for short-lived cells,
although stem cells are scanty and
differentiate rarely (more commonly in
response to injury)
Life span Short, usually only days
Differentiation Asymmetric division of stem cells
gives rise to new stem cells and
progenitor cells that divide
sequentially and differentiate
into mature cells
High, usually associated with complete
Frequent, apparently due to high
content of stem and progenitor cells
Minimal. Differentiated cells have a
too-short life span to accumulate
substantial amounts of damaged structures
(waste materials). Stem and progenitor
cells do not accumulate damaged structures
either, because the latter are efficiently diluted
by cell divisions
Regeneration capacity Low, usually associated with incomplete
regeneration, resulting in scarring
Rare, apparently due to low content of
stem and progenitor cells
Pronounced. Differentiated cells have long
life spans, resulting in progressive
accumulation of waste materials
[e.g., lipofuscin, senescent (giant)
mitochondria, and aberrant proteins]
aA number of postmitotic cell types, such as mature hepatocytes or fibroblasts, show intermediate renewal and age-related characteristics
and, thus, cannot be ascribed to any of the two groups.
bRefers to stem and progenitor cells giving rise to differentiated cells.
MITOCHONDRIAL TURNOVER AND AGING505
and lysosomal features of importance for aging, the inter-
play and cross-talk between mitochondria and the lyso-
mitochondrial–lysosomal axis theory ofaging(30).In essence,
this hypothesis suggests that depressed macroautophagy
secondary to the accumulation of lipofuscin inside the lyso-
somal compartment results in prolongation of mitochondrial
life span with accumulation of enlarged functionally effete
mitochondria, and ensuing decline in ATP production, in-
creased formation of ROS, accelerated formation of lipofuscin
and, finally, lysosomal labilization with activation of the ap-
optotic or necrotic pathways. These alterations at the cellular
level inevitably lead to progressive functional decline, de-
creased adaptability, and an increased probability of disease
and death for the organism.
II. ROS, Mitochondrial Damage, and Aging
A. Biomolecular damage under normal conditions
Soon after the important discovery that ROS, including the
superoxide anion radical (O2
both of which are short-lived with half-lives of 10?6and 10?9
seconds, respectively, form within living cells as a conse-
quence of normal respiration (94), Denham Harman (98)
postulated that biologic aging (senescence) occurs because of
the accumulation of oxidatively damaged macromolecules.
This theory, called ‘‘the free radical theory of aging,’’ although
initially poorly accepted, has gathered an increasing number
of followers over time as more supporting evidence has been
presented. Today, the role of free radicals as important con-
tributors to aging is considered most likely, and extensive
studies on various biologic species ranging from yeast to
humans are in support, although a final confirmation is still
lacking (10, 87, 167, 200, 213). The free radical theory of aging,
which points to an intrinsic mechanism underlying age-
related molecular damage, does not in any way exclude that
other factors may also be involved in the aging process (e.g.,
evolution, somatic mutations, errors in protein synthesis, ac-
cumulation of waste products, neuroendocrine and immu-
nologic disturbances). The possibility that many mechanisms
may contribute to the aging process is reflected in the existing
numerous theories of aging (some of them having only his-
toric value), which are systematized in a number of reviews
(156, 187, 255).
The process of cellular respiration is tightly associated with
the electron-transport chain and the transfer of electrons from
substrates (e.g., NADPH from complex II) to the final acceptor
(molecular oxygen) in complex IV. The electron transport is
associated with translocation of protons from the mitochon-
drial matrix to the mitochondrial intermembrane space,
which originates a membrane potential. This potential is
coupled with phosphorylation of ADP to form ATP at com-
plex V. Both free radicals and other ROS form continuously
because of unavoidable electron leakage from mitochon-
drial complexes during electron transport and reductive one-
electron transfer processes in the cytosol. The addition of one
electron per oxygen molecule yields the superoxide anion
peroxide, may be toxic to some enzymes, particularly mito-
chondrial aconitase (261). Most superoxide is, however, con-
verted to hydrogen peroxide by superoxide dismutases
?–) and hydroxyl radical (HO?),
?–, which in itself, or after dismutation to hydrogen
(SODs). Mammalian cells contain cytosolic Cu, Zn-SOD,
whereas mitochondria contain Mn-SOD in their matrix as
well as Cu, Zn-SOD in their intermembranous space (81). An
extracellular form of SOD exists. Although the dismutation of
superoxide to hydrogen peroxide is by itself a very rapid
spontaneous process, catalysis by SODs increases the rate of
superoxide dismutation to hydrogen peroxide and oxygen
*1,000-fold. Once hydrogen peroxide is formed, it is rapidly
transformed into water. In the mitochondrial matrix, this
takes place mainly by the peroxiredoxin=thioredoxin system
glutathione peroxidase and catalase work in concert in the
cytosol to degrade hydrogen peroxide.
regulates most cytosolic redox activity (220). However, if not
eliminated, it can also react with Fe(II) during Fenton-type
reactions, resulting in the formation of the very reactive hy-
droxyl radical. In addition, superoxide can directly reduce
Fe(III) to Fe(II), which further contributes to the creation of
HO?or the likewise reactive ferryl or perferryl radicals. All of
these radicals attack surrounding biomolecules (i.e., nucleic
in direct relation to Fe(II) catalysis), resulting in damage to
biomolecules with attached low-mass iron (94). Although
most of the hydrogen peroxide is eliminated by glutathione
peroxidase and catalase, some of it remains and may diffuse
for some distance (e.g., to the lysosomal compartment, which
lacks hydrogen peroxide–degrading enzymes). Because ly-
sosomes not only lack these enzymes, but also are rich in
reactive iron as a consequence of the degradation of ferrugi-
nous materials (see Section VI.A), the formation of these
radicals takes place mainly inside these organelles. This may
result in lysosomal rupture, followed by damage to cytosolic
structures as well as to nuclear and mitochondrial DNA as a
result of the relocation of redox-active iron and hydrolytic
enzymes (64, 127).
It may be assumed that the reason nature has found it
necessary to speed up by 1,000 times the already rapid
is that the capacity of superoxide to reduce Fe(III) to Fe(II) is a
very dangerous one, allowing the formation of hydroxyl
radicals if hydrogen peroxide is available (see earlier). Basic
metabolic pathways involved in ROS production are sche-
matically presented in Fig. 1.
Although ROS formation apparently is the main source
of oxidative damage, it is not the only one. Another im-
portant damaging mechanism is glycation [i.e., a reaction of
glucose and other reducing sugars with protein amino
groups, resulting in the formation of advanced glycation end
products (AGEs)]. AGEs, which can bind to DNA and pro-
teins, may in turn induce mutations and protein–protein
cross-linking. The latter phenomenon is of special impor-
tance extracellularly and can cause stiffening of elastic tis-
sues in the skin, vessels, and the eye lens (25, 82, 134). In
addition, the reactive metabolite, S-adenosylmethionine, can
methylate guanine, affecting the hydrogen-bonding ability
of DNA bases.
Furthermore, because of their inherent instability, many
macromolecules can undergo spontaneous modifications (not
and depurination, deamination of DNA bases, isomerization,
racemization, and deamidation of protein amino acid resi-
506 TERMAN ET AL.
dues, or dephosphorylation of phosphoproteins [reviewed
B. Imperfect turnover of damaged biologic structures
Oxidatively or otherwise damaged biologic structures are
either repaired (e.g., single bases in DNA molecules are re-
placed) or degraded and completely replaced by newly syn-
thesized structures, as is the case for proteins, organelles, and
whole cells. Proteins, predominantly short-lived ones in the
nucleus and cytosol, are degraded mainly by calpains and
proteasomes, whereas most long-lived proteins and all or-
ganelles are digested in the lysosomal compartment in the
long been known that proteins intended for degradation by
proteasomes have to be tagged by ubiquitin, but it is now
recognized that some ubiquitinized proteins also are de-
systems for degradation can compensate for each other (177,
242). Mitochondria possess their own proteolytic system,
which includes Lon, Clp-like proteases, and AAA proteases
(see Section IV). Irreversibly damaged cells are removed
by self-killing programs, including apoptotic (caspase-
dependent) programmed cell death (PCD-I), autophagic cell
death (PCD-II), or, occasionally, necrosis (PCD-III) (71). Cel-
lular catabolic pathways are summarized in Table 2.
mainly in mitochondria as a result of electron leak from the electron-transport chain and to a lesser extent in the cytosol,
because of the activity of one-electron transfer oxidases and the cytochrome P450 system. Superoxide rapidly dismutates
spontaneously to hydrogen peroxide (H2O2), but this reaction is further increased 1,000-fold by mitochondrial and cytosolic
forms of superoxide dismutase (SOD). This indicates that superoxide is a dangerous molecule, probably because of its
capacity to reduce Fe(III) to Fe(II). Hydrogen peroxide, an uncharged molecule, diffuses freely within the cell. Most hydrogen
peroxide is eliminated by cytosolic and mitochondrial glutathione peroxidase (GPX), as well as by catalase in peroxisomes. In
the presence of redox-active iron, hydrogen peroxide is homolytically cleaved under the formation of highly reactive hy-
droxyl radicals (HO?; the Fenton reaction). Hydroxyl radicals can damage a variety of biomolecules, including nucleic acids,
proteins, and lipids. By reacting with polyunsaturated fatty acids, they initiate a chain reaction, resulting in the formation of
aldehydes that can cause additional macromolecular damage. The reaction between superoxide and nitric oxide (NO?,
formed from l-arginine in the presence of nitric oxide synthase, NOS), produces peroxinitrite (ONOO?), which can generate a
hydroxyl radical at acidic pH (e.g., in the lysosomal compartment). This possibility is provided by the fact that nitric oxide
(which is uncharged and thus passes biologic membranes) can diffuse into the lysosomes, where it may react with superoxide
derived from autophagocytosed mitochondria that are under degradation. Continuous arrows, transformation; dashed arrows,
diffusion of substances.
Metabolic pathways involved in the production of cellular ROS. Superoxide anion radicals (O2
??) are produced
MITOCHONDRIAL TURNOVER AND AGING507
The renewal of long-lived postmitotic cells, which are
poorly (or not at all) replaced through division and differen-
tiation of stem cells, is practically fully dependent on intra-
cellular degradation pathways. As pointed out earlier, the
latter do not function perfectly and, as a result, damaged
structures (such as defective mitochondria, lipofuscin-loaded
lysosomes, and oxidized proteins) progressively accumulate
in time, resulting in a diminished amount of normal cellular
structures.Thiswillmakethefunction ofthe cellslessefficient
and decrease their adaptability. The accumulation of biologic
garbage also is associated with certain toxic effects, such as
increased ROS production by senescent mitochondria, or en-
hancement of oxidative stress with release of lysosomal en-
zymes by lipofuscin-loaded lysosomes (see Section VI). These
changes result in progressive functional decline of postmitotic
cells, such as neurons, cardiac myocytes, and skeletal muscle
fibers, making an aged organism fragile and unable to with-
individual is thus reflected on the cellular level.
Consistent with the idea that aging is largely dependent on
the ultimate degeneration of long-lived postmitotic cells, a
primitive cnidarian animal, Hydra vulgaris, has been shown to
escape aging for 4 years in a controlled laboratory environ-
ment (150). The most plausible explanation for the absence of
aging in hydra is that this animal, as well as other cnidarians,
totally lacks long-lived postmitotic cells. All cells of cnidarian
animals are continuously replaced through the division and
differentiation of interstitial stem cells. Interestingly, all
higher animals, which evolved later than cnidarians, contain
postmitotic cells, and therefore have limited life spans. It is
possible that the appearance of long-lived postmitotic cells, in
particular, long-lived neurons, came along evolutionarily
because this trait was associated with certain advantages,
providing for better evolutionary fitness. Cnidarians are
known to possess a primitive nervous system, consisting of a
network of dissociated short-lived neurons. These animals
can react only nonspecifically on external stimuli and do not
develop conditioned responses. In contrast, higher animals
have a more-developed nervous system, consisting of long-
lived postmitotic neurons, allowing conditioned responses,
and consequently, providing better adaptation to their envi-
ronment. Apparently, the presence of long-lived neurons
promoted the development of long-term memory, associated
with conditioning. The price for this better evolutionary fit-
ness was, however, a limited life span because of unavoidable
postmitotic cellular aging. This hypothesis is described in
detail elsewhere (234).
C. Major targets of ROS attack:
mitochondria and lysosomes
As the main sites of endogenously generated ROS, mito-
chondria are the logical and major targets of ROS attack,
which, in combination with the insufficient degradation and
replacement of damaged mitochondria, results in their pro-
nounced alterations with age. The mitochondria of aged
postmitotic cells are usually enlarged, sometimes enormously
so (and are occasionally called giant mitochondria), and are
functionally insufficient (see also Section VII). Abnormal mi-
tochondria of aged cardiac myocytes are shown in Figs. 2
The role of mitochondria in aging is reflected in the re-
finements of the free radical theory of aging that stresses the
importance of mitochondrial ROS production and oxidative
damage to mitochondrial components in the overall role of
ROS in aging (57, 99). Mitochondrial proteins are affected not
only because of direct oxidative damage, but also as a con-
sequence of oxidant-induced mutations in the mitochondrial
DNA (mtDNA) that codes for 13 proteins involved in oxida-
tive phosphorylation (53). Some changes in mitochondrial
proteins arise from damage to the nuclear DNA. These
changes would apparently affect all cellular mitochondria,
whereas mtDNA mutations would change only a portion of
mutated DNA (see Section VII.C). It is believed that the
properties of mtDNA, which is a circular bacterial type not
protected by histones, probably has a poor repair capacity
compared with nuclear DNA, thereby contributing to its high
vulnerability to ROS (176). Although recent findings suggest
that the repair mechanisms of mtDNA are more advanced
than originally thought (66), age-related damage to mtDNA is
well established (176).
Homozygous knockout mice expressing defective mtDNA
polymerase provide strong evidence for the role of mito-
chondrial damage in aging (244). These animals show a dra-
matically increased rate of mtDNA mutations and are
characterized by premature development of age-related
phenotypic alterations of various organs, as well as by re-
Table 2. Cellular Degradation Processes
Degradation processLocation Enzymes involvedTargets
Cytosolic proteolysisCytosol Calpains, proteasomesCytosolic proteins (mainly
Mitochondrial proteins Mitochondrial proteolysisMitochondriaLon, Clp-like, and
Acid hydrolases Autophagy (macroautophagy,
Programmed cell death (PCD)
LysosomesAll cytosolic macromolecules
Whole cell Effector caspases, lysosomal
cathepsins, and endonucleases
in PCD-I (classic apoptosis);
acid hydrolases in PCD-II
(autophagic cell death) and
PCD-III (programmed necrosis)
All cellular components
508TERMAN ET AL.
duced life span. A later publication from the same group (243)
showed that these prematurely aging mice having defective
mtDNA polymerase produce normal amounts of ROS, which
seems to be in opposition to the free radical theory of aging.
However, considering that, in normal aging, damage to
mtDNA occurs secondary to ROS production, it is predictable
that the induction of mitochondrial damage in a different way
(e.g., by disturbing the function of mtDNA polymerase) may
as well result in the development of senescence-like alter-
ations. It should be also kept in mind that because oxidatively
damaged structures (including defective mitochondria) ac-
cumulate because of insufficient clearance mechanisms, the
increase of oxidative stress is not a necessary requirement
for aging to occur. Damaged structures would accumulate
anyway, although with a lower rate, at constant or even
decreasing levels of oxidative stress.
has been somewhat challenged by the finding that Mclk1þ=?
damage in neonatal rat car-
diac myocytes. (A, B) Con-
focal laser scanning images
(488-nm excitation) of form-
aldehyde-fixed cells aged 1
and 4 weeks, respectively. Lf,
lipofuscin granules. (C, D)
excitation) of cardiac myo-
cytes (aged 17 days and 3
months, respectively) vitally
stained with mitochondrial
tracker JC-1. Note the abun-
dant enlarged ‘‘green’’ mito-
chondria with low membrane
potential (thin arrows) and a
lesser amount of slender ‘‘red’’
membrane potential in (D)
versus (C). (E, F) The 17-day-
inhibitor 3-methyladenine for
12 days, contain some en-
arrows), as well as prominent
chondria (thick arrows), many
of which show a low mem-
brane potential. Bar, 10mm.
MITOCHONDRIAL TURNOVER AND AGING509
mice, with a reduced activity of a mitochondrial enzyme
necessary for ubiquinone synthesis, were characterized by
increased hydrogen peroxide production and elevated
protein carbonyl levels (indicative of protein oxidation) in
hepatocyte mitochondria, but still lived longer than the
wild-type animals (133). The Mclk1þ=?mutants, however,
showed reduced carbonyl and isoprostane levels in the
nonmitochondrial cytoplasmic compartments, suggesting
decreased oxidative damage to proteins and lipids. The pos-
itive changes in the nonmitochondrial part of the cytoplasm,
probably involving lysosomal proteins and lipids, may to
some extent explain this paradoxic finding. Another possible
explanation of these results may be that an enhanced pro-
duction of mitochondrial ROS induces upregulation of stress
proteins, such as HSP70, which, after autophagy, reduce the
concentration of lysosomal redox-active iron. This in turn
would depress lysosomal formation of lipofuscin and prevent
failing autophagy and reduced cellular ‘‘self-cleaning’’ (126,
out that the importance of these-mentioned results for the
understanding of the free radical theory of aging is dimin-
ished by the fact that the oxidative-stress parameters were
assessed in hepatocytes, which are much less affected by age
than arelong-lived postmitoticcells, suchascardiacmyocytes
in the synthesis of heme (including that of the mitochondrial
inner membrane protein cytochrome c) and most iron–sulfur
clusters (199). It is possible that some mitochondrial iron is in
reactive form and able to support Fenton-type reactions. If so,
the combination of redox-active iron with internally formed
ROS would contribute to mitochondrial damage.
For the same reason, lysosomes are also sensitive to oxi-
dative stress, leading to a gradual accumulation of the in-
tralysosomal indigestible material, lipofuscin, paralleled by a
decline in the lysosomal degradative function (30). However,
chondrial changes associated
with aging and inhibition of
autophagy. (A, B) Electron
rat cardiac myocytes cultured
The aged cells contain en-
larged (giant) mitochondria
dense matrix. (C, D) The 17-
day-old cardiac myocytes, ex-
posed to 3-methyladenine for
small, as well as some large
(compare with Fig. 2). M,
mitochondria; Lf, lipofuscin.
510TERMAN ET AL.
compared with the generally accepted role of mitochondrial
decay in aging, the roles of lysosomal malfunction and the
cross-talk between lysosomes and mitochondria in aging re-
main less recognized.
The progress of age-related mitochondrial degeneration is,
to a large extent, dependent on the failure of mitochondrial-
turnover mechanisms, including (a) mitochondriogenesis or
the generation of more mitochondrial mass, (b) mitochondrial
fusion and fission, (c) the monitoring of protein folding and
assembly by molecular chaperones and energy-dependent
proteases, and (d) the removal of severely damaged mito-
chondria by autophagy (119). These mechanisms, as well as
their age-associated malfunction, are described more in detail
III. Mitochondrial Fusion, Fission, and Biogenesis
A. The role of mitochondrial dynamics
fusing and dividing (Fig. 4). The harmonious balance of these
two opposing processes is responsible for the prevailing
morphologic features of mitochondria, their distribution, in-
heritance, and function (61).When the mitochondrial fusion is
blocked, the normal tubular network of mitochondria trans-
forms into fragmented mitochondria (40, 41, 90,173), whereas
the blocking of the opposing process, mitochondrial fission,
results in elongated, interconnected mitochondrial tubules
(211, 217, 263). Likewise, the overexpression of fusion pro-
teins, such as mitofusin 2 (Mfn2), results in the formation of
large mitochondria or long mitochondrial tubules, whereas
the overexpression of fission proteins, such as the dynamin-
related protein 1 (Drp1) or mitochondrial fission protein 1
(Fis1), results in the formation of small fragmented mito-
Once the delicate balance between fusion and fission is lost,
notonlyisthe morphology altered,butvarious mitochondrial
and cellular functions are changed as well. When fusion is
decreased, as in Mfn-null or OPA1 (optic atrophy protein 1)-
depleted cells, fragmentation of the mitochondrial network is
followed by reduced glucose oxidation, decreased mito-
chondrial respiration, and diminished mitochondrial mem-
brane potential (179). This process is reversible if respiration
can be restored by the reintroduction of the affected proteins
Mitochondrial fusion and fission is also intimately associ-
ated with mitochondria-mediated apoptosis. (178). At least
two proteins involved in mitochondrial fusion appear to
protect cells from apoptosis (112) by controlling the re-
modeling of mitochondrial cristae (47, 80). Mitochondrial
fission resulting from the overexpression of Drp1 protects the
cells against Ca-mediated apoptosis by interrupting in-
tramitochondrial Ca2þwaves and reducing overall mito-
chondrial Ca2þuptake (228). Conversely, a different fission
protein, hFis1 (human homologue of Fis1), seems to promote
apoptosis (108, 140).
Last, mitochondrial fusion and fission is related to the en-
ergetic sources of the cell. When oxidative phosphorylation is
compromised (e.g., when glycolysis is the main source of
ATP), mitochondria appear punctuated or vesicular. Con-
versely, when glycolysis is blocked, tubular mitochondria
form (16). These observations do not seem to conform to
the findings that senescent cells accumulate enlarged mito-
chondria with decreased inner membrane potential and
consequent decreased ATP production (169). Apparently,
mechanisms that control mitochondrial morphology de-
pending on cellular energetic status are different from those
involved in age-related mitochondrial alterations (see Section
VII). One should not underestimate the intimate relation be-
tween mitochondrial structure and bioenergetics. This topic is
reviewed in an excellent way by Benard and Rossignol (16).
tochondria with different membrane potentials. (A) GFP
fluorescence identifies mitochondria. TMRE (tetramethyl-
rhodamine ethyl ester) is used to calculate the membrane
potential. The pseudocolor images at the bottom are used to
identify the initial and daughter mitochondria. (B) Average
membrane potential before (left, gray) and after fission (right,
solid and empty circles denote depolarized and hyperpolarized
mitochondria, respectively) are shown. Reprinted from Twig
et al. (246), with permission from Macmillan Publishers Ltd.
(For interpretation of the references to color in this figure
legend, the reader is referred to the web version of this article
Mitochondrial fission leads to two daughter mi-
MITOCHONDRIAL TURNOVER AND AGING 511
In this section, we discuss mitochondrial fusion, fission,
and biogenesis, basic processes involved in mitochondrial
dynamics, dysregulation of which may be responsible for
changes in mitochondrial turnover that accompany aging(see
further Section VII). Mechanisms of mitochondrial fusion and
fission are schematically presented in Figs. 5 and 6.
B. Mitochondrial fusion
Mitochondrial fusion resembles virus-mediated fusion
or SNARE-dependent (soluble N-ethylmaleimide–sensitive
factor-attachment receptor) membrane fusion, but is unique
in the sense that it requires the coordinated fusion of the inner
andouter mitochondrial membranesand ismore complicated
than was initially thought (38).
The core components of the mitochondrial fusion machin-
ery were first identified in Drosophila and yeast. Fuzzy onion
(Fzo) protein and its yeast counterpart Fzo1 were the first
proteins shown to play a role in mitochondrial fusion (93).
This discovery ignited studies of mitochondrial fusion and
fission in mammalian systems, in which the key players are
proteins with a largely conserved genetic background, work
that resulted in the identification of several mammalian or-
thologues. Fusion proteins in mammals are typically large
GTPases localized in the mitochondrial membranes. Figure
5A illustrates key players in the fusion process. For example,
Mfn1 (mitofusin 1) and Mfn2 are closely related mammalian
homologues, which play an important role in the fusion of the
outer mitochondrial membrane (40, 41). They contain long
transmembrane domains with both the C and N termini
protruding from the outer membrane into the cytosol and
require low levels of GTP and mitochondrial membrane po-
tential formitochondrial fusiontooccur(195).AlthoughMfn1
is believed to be involved primarily in mitochondrial tether-
ing, Mfn1 and Mfn2 play similar roles and can, under certain
conditions, function interchangeably. Cells underexpressing
Mfn1 can restore their mitochondrial fusion activity by
overexpressing Mfn2, and vice versa. It has been shown that
the requirements for the expression of either of the mitofusins
are tissue dependent (104).
OPA1 is another member of the mitochondrial-fusion
protein family, and it was initially identified as a protein in-
volved in dominant optic atrophy, an autosomally inherited
disease resulting in vision loss (2). OPA1 is localized in the
intermembrane space and is associated with the inner mito-
chondrial membrane (173). The role of OPA1 remains largely
unknown, but it may be directly involved in inner membrane
fusion, controlling the shape and structure of cristae. Over-
expression of OPA1 can result in either mitochondrial frag-
mentation or elongation, depending on the experimental
model used (40). It also can cause mitochondrial fragmenta-
tion while retaining its fusion activity, making it unique
among mitochondrial fusion proteins. These clues point to
the special role that OPA1 plays in mitochondrial fusion.
It has been suggested that regulation of the OPA1 function
may be due to posttranslational modifications of this protein
Mfn, and the inner-membrane protein, OPA1, regulate mitochondrial fusion in mammalian cells. PARL cleaves the trans-
membrane domain of OPA1 and activates it, which may result in oligomerization and assembly in large complexes responsible
for mitochondrial remodeling and cristae junction formation. (B) Mitochondrial fission. Drp-1, the key component of mito-
chondrial fission, is localized in the cytoplasm, but may be translocated to the mitochondrial outer membrane, triggered by
an unknown signal, where it binds to other proteins and forms large circular complexes. These complexes then send signals to
and from the inner membrane to coordinate the fission of both membranes, which is believed to be regulated by Rab32 and
PKA. X, unidentified factors involved in fusion and fission. A complete description of the processes is given in (20).
Schematic models of mitochondrial fusion and fission. (A) Mitochondrial fusion. The outer membrane protein,
512 TERMAN ET AL.
or to the regulation of its function by mitochondrial prote-
Impaired fusion capacity of depolarized or otherwise
damaged mitochondria has been reported (106, 141, 147, 152,
157, 159). It is, then, not surprising that such mitochondria
often show OPA1 degradation, which probably underlies an
impaired fusion and any subsequent exchange of mitochon-
drial components (69, 105, 141, 158, 214). A decreased OPA1
expression is one of the apparent reasons for the inability of
senescent-like enlarged mitochondria to fuse and exchange
their contents with normal mitochondria (see Section VII).
Fusion may be necessary for maintaining and restoring mi-
tochondrial function by facilitating the stochastic redistribu-
tion of soluble and membrane components of normal and
defective mitochondria (38, 41, 61, 174). Indeed, clear evi-
dence indicates that the mixing of mitochondrial components
occurs after fusion (6, 34, 113). However, the ability of mito-
chondria to fuse may be hampered above a certain threshold
of mitochondrial damage, suggesting that mitochondrial de-
fects, including age-related ones (Section VII), cannot be
completely eliminated by fusion with functionally normal
C. Mitochondrial fission
Mitochondrial fission is the functional counterpart of fu-
sion, but very little is known about its underlying molecular
mechanisms. Drp1 is the key component in this process (see
Fig. 5B). It is expressed largely in the cytosol, but also forms
punctuate expression foci in the inner mitochondrial mem-
brane (211). The punctuate expression of Drp1 determines the
location of future fission sites on mitochondrial tubules. As in
the case of mitochondrial fusion, many insights into the
mechanism of mitochondrial fission have been derived from
yeast studies. Drp1 is also a member of the GTPase family,
which means that it requires GTP to initiate the constriction of
mitochondria at the fission sites. In addition, other cellular
processes, such as the organization, division, and distribution
of mitochondrial DNA, rely on the functional fission ma-
chinery. Fis1 is another component of the fission machinery
fusion and fission. For mitochondrial fusion to occur, mitochondria must be in close contact. It also requires mitochondrial
fusion proteins (see Fig. 5A for details), functional mitochondrial inner membrane potential, and low concentrations of GTP.
(B) Fusion of two mitochondria labeled with different fluorescent proteins (e.g., GFP and DsRed2) results in the formation of a
single mitochondrion with intermixed mitochondrial contents of the parent mitochondria. Giant mitochondria do not appear
to fuse with normal mitochondria or with each other. (C) A fluorescence-microscopy image of a polykaryon formed by fusion
of cells containing mitochondria labeled with GFP and DsRed2. Four hours after fusion, most mitochondria have fused with
others and exchanged their mitochondrial matrix components, containing both fluorescent labels appearing as a yellow color
in the composite image. The nuclei were counterstained with DAPI (blue). (D) A cell containing giant mitochondria labeled
with mitochondria-targeted GFP were fused with other cells containing normal mitochondria labeled with DsRed2. Giant
mitochondria remain single-labeled even 8h after fusion, whereas normal mitochondria show colocalization of both fluo-
rescent probes. (C*, D*) Enlarged sections of the images (C) and (D), respectively, with both the composite image and green
and red components of the same image. Reprinted from Navratil et al. (169) with permission from Elsevier. (For interpretation
of the references to color in this figure legend, the reader is referred to the web version of this article at www.liebertonline
Mitochondrial fusion in mammalian cells. (A) Mitochondrial morphology is controlled by two opposing processes,
MITOCHONDRIAL TURNOVER AND AGING 513
responsible for the recruitment of Drp1 into mitochondria.
Unlike Drp1, it is uniformly expressed in the outer mito-
chondrial membrane, with most of the protein facing the cy-
tosol (108). Other proteins that are thought to play a role in
mitochondrial fission include endophilin B1, mitochondrial
protein 18 (MTP18), ganglioside-induced differentiation-
associated protein (GDAP), mitochondrial rho- (Miro), and
mitochondrial rho-2 (Miro-2) proteins (223). Unlike mito-
chondrial fusion, fission does not require a regular membrane
potential because it can be induced by a collapse of the
membrane potential resulting from ATP depletion due to the
inhibition of ATP synthase or Na=K ATPase (180).
Fission appears to aid in the elimination of damaged mi-
tochondria, thereby delaying the onset of age-related dam-
age. Recent findings indicate that fission is useful for the
segregation of irreversibly damaged, depolarized, fusion-
incompetent mitochondria, as well as for their subsequent
elimination by autophagy, as is illustrated in Fig. 7 (73, 182).
S-nitrosocystein) results in fragmentation and the accumula-
tion of mitochondria in autophagosomes (12). Most surpris-
ingly, commonly fusion triggers fission that produces two
metabolically different daughter units. Such daughter mito-
chondria have different membranous structures (12), their
DNA is not equally redistributed (6, 12), and one of the two in
the pair has a reduced membrane potential. The depolarized
daughter mitochondria are less likely to re-fuse and are more
likely to become autophagocytosed (246).
Although the molecular mechanisms behind the segrega-
tion of defective mitochondrial components through asym-
metric fission are in need of further investigation, these
important findings have already contributed to the under-
standing of mitochondrial changes associated with aging.
D. Mitochondrial biogenesis
Mitochondriogenesis, or mitochondrial biogenesis, is a vi-
tal process in mitochondrial turnover. It is believed to involve
*1,000 genes and to affect 20% of the cellular proteins (4, 79,
136, 137, 168, 260). As nonfunctional mitochondria are elimi-
nated by autophagy, mitochondrial biogenesis is needed to
sustain energy production and physiological homeostasis in
the cell. Factors that regulate mitochondrial biogenesis in-
clude the levels of nutrients available, the presence or absence
of hormones, temperature, exercise, hypoxia, stress, and ag-
ing. Mitochondriogenesis has been shown to decrease with
age (see Section VII), contributing to progressive mitochon-
drial decay (143). We later present a summary of the role of
various factors involved in mitochondrial biogenesis.
Thyroid and steroid hormones regulate the expression of
of mitochondrial proteins. An increase in the levels of the
thyroid hormone thyroxine causes hyperplasia and increases
the number and mass of mitochondria in liver and cardiac
muscle (88, 259). Steroid hormones also affect mitochondrial
biogenesis in adipose tissue by either inhibiting (testosterone)
or promoting (progesterone) the expression of transcription
factors involved in this process (193).
According to Lo ´pez-Lluch et al. (143), the transcription
factors involved in mitochondrial biogenesis belong to three
groups: ubiquitous transcription factors, nuclear respiratory
factors, and coactivators. Among the latter group, the tran-
scription factor most important to mitochondriogenesis is the
proliferator-activated receptor g coactivator-1a (PGC-1a)
(143). Although the contribution of the various factors af-
fecting mitochondrial biogenesis cannot be separated at the
present time, PGC-1a has emerged as the common intracel-
extracellular material by in-
thereby forming early endo-
acidified late endosomes. The
enzymes by fusion with secre-
tory vesicles from the trans-
Golgi network (TGN). Further
maturation leads to lysosome
formation. Cytosolic macro-
molecules may be directly
engulfed by invaginations of
the lysosomal membrane (mi-
croautophagy), whereas or-
ganelles (e.g., mitochondria)
are being enclosed by a newly
formed phagophore, resulting
in the formation of an autop-
which then fuses with either a
late endosome or a lysosome
(or, perhaps, with secretory
vesicles from the TGN), forming an autophagolysosome. Certain proteins are delivered to lysosomes with the help of
chaperones, such as Hsp73 (chaperone-mediated autophagy).
514 TERMAN ET AL.
lular mediator for mitochondrial biogenesis. PGC-1a acts by
coordinating and modulating the activity of other transcrip-
tion factors (e.g., nuclear transcription factors such as the
nuclear respiratory factor 1, NRF-1, peroxisome proliferator–
activated receptors such as PPARa, and the mitochondrial
transcription factor mtTFA) that are involved in mitochon-
drial biogenesis (184), or by interacting at DNA sites, where it
promotes the recruitment of additional coactivators such as
the steroid receptor coactivator-1 (SRC-1), which itself alters
the DNA structure to make it more available for processing
by the transcriptional machinery (183).
The number of known activators of PGC-1a is extensive
(143). They include nitric oxide, the cAMP response element–
binding protein (CREB), and AMP-activated kinase (AMPK).
The latter is a cellular energy sensor that links mitochon-
driogenesis to several aging-associated processes such as in-
sulin resistance, obesity, and decreased fatty acid catabolism.
activate PGC-1a. NO regulates PGC-1a both directly and in-
directly (14, 131). Direct regulation by NO occurs through
upregulation of transcription factors by cyclic GMP, which in
turn leads to the expression of sirtuins [silent information
regulation 2 (Sir2) proteins] that deacetylate PGC-1a, making
it more active (24, 172). Other compounds such as resveratrol
also activate SIRT1 (sirtuin homologue 1) and cause PGC-1a
to increase in hepatocyte and myocyte systems (14, 131).
A few reports described negative regulators of PGC-1a.
These regulators include RIP140, which interacts with the
nuclear receptors (e.g., retinoic X receptor) blocking the in-
teractions of such receptors with PPAR receptors (257). Other
negative regulators include p160 myoglobin-binding protein,
glucose, GC5N acetyl transferase, and a mutant form of
Huntingtin (143). The presence of both positive and negative
regulators of PGC-1a is evidence of a complex system that
coordinates mitochondrial biogenesis with the energetic de-
mands of the cells.
IV. Mitochondrial Proteolytic Systems
To maintain active mitochondria, cells are equipped with
As pointed out later, mitochondrial autophagy is a mecha-
nism for getting rid of severely damaged organelles. The ad-
vancement of mitochondrial damage can, however, also be
lessened by the proteolytic machinery of the mitochondria,
involving the ATP-dependent matrix proteases that are sub-
divided into the Lon (LonA and LonB subfamilies), Clp-like,
and AAA proteases. The existence of this proteolytic system
extends the half-life of mitochondria, apparently by decreas-
ing demands for the more energy-consuming autophagic
Only Lon proteases are known to eliminate oxidatively
modified proteins (21, 32, 114, 148), whereas the physiologic
functions of the Clp-like and AAA proteases are still less well
by the fact that the downregulation of human LonA proteases
results in apoptotic cell death within a few days (22), whereas
a less-dramatic downregulation results inthe accumulation of
the same type of large and malformed mitochondria that are
normally found in aged postmitotic cells. These findings
suggest a strong correlation between aging and Lon protease
The Lon proteases are encoded by nuclear DNA and are
composed of three phylogenetically well-conserved domains
(107, 164). The N domain interacts, together with the middle
one (called AAAþ), which also binds ATP, with the protein to
be degraded, whereas the third domain (P) contains the active
mitochondrial DNA and are released and activated by oxi-
dative stress (31, 138, 145). When conditions for oxidative
damage to mitochondrial proteins emerge, it is possible that
mitochondrial degradation ensues.
Lon-like proteolytic activity has been found to be consid-
erablyinhibited oreven inactivated in oldrats(7) andmore so
in postmitotic cells than in replicating ones. Mitochondria
from old mice contain increased amounts of oxidized pro-
teins, especially aconitase, reflecting an age-related decrease
by ROS. Interestingly, aconitase inactivation is significantly
less pronounced in CR animals and in animals exposed to the
CR-mimetic drug resveratrol. This polyphenol, like many
other plant-derived compounds with supposed antioxidant
activity, is also an iron chelator (authors’ unpublished ob-
servations), which suggests that it may act by chelating
Fe(II) into a complex where iron is nonredox active, thereby
preventing the formation of hydroxyl radicals through
Fenton-type reactions. The well-known time-dependent ac-
cumulations of iron and other heavy metals in postmitotic
cells (129) may be an important factor behind the inactivation
of Lon proteases that is found in aged individuals. This hy-
pothesis about the toxic effects of accumulated iron is fur-
ther supported by the mitochondria-revitalization effect of
another iron chelator, N-tert-butyl hydroxylamine (252).
V. Mitochondrial Turnover by Autophagy
A. The main functions of the lysosomal compartment
The lysosomal compartment is crucial for cell maintenance
and has a variety of important functions, including endocytic
uptake of materials from the outside and autophagic degra-
dation of damaged mitochondria and other organelles, such
as ribosomes, endoplasmic reticulum, and the proteasome
microorganelles, as well as numerous, mostly long-lived,
proteins (see Table 2). Consequently, lysosomes exist in all
kinds of plant and animal cells, except erythrocytes, which
have a very specialized function and a minimal turnover of
their constituents. Inside the lysosomal compartment, the
degradation of endocytosed or autophagocytosed materials
takes place in an acidic environment (pH * 4–5), which is
maintained by ATP-dependent proton pumps present in the
lysosomal membrane. Such pumps also are present in the
plasma membrane, especially in tumor cells, in which anaer-
obic glycolysis results in significant production of lactic acid
(the Warburg effect). These pumps are then needed for the
254). Consequently, many tumor cells, especially the highly
malignant and rapidly growing ones, surround themselves
with a microenvironment with a low pH.
After synthesis in the endoplasmic reticulum, lysosomal
hydrolases are tagged with mannose-6-phosphate (MP) at the
cis-Golgi area and then enclosed in transport vesicles (some-
times named primary lysosomes, although they have a neu-
tral pH) in the trans-Golgi network (TGN) with the help of
MP receptors. The vesicles containing the newly produced
MITOCHONDRIAL TURNOVER AND AGING 515
hydrolases are then transported to slightly acidic (pH*6) late
endosomes, which arise from early endosomes containing
endocytosed material. The lysosomal hydrolases are then
to the Golgi apparatus. Finally, the late endosomes mature to
lysosomes that lack MP receptors, are rich in acid hydrolases,
have a pH of 4–5, and contain material to be degraded.
The acidic lysosomal compartment contains a wide spec-
trum of hydrolytic enzymes, which play a major role in the
intracellular recycling of proteins, polysaccharides, phos-
pholipids, and other biomolecules. Lysosomal proteases (ca-
thepsins) are apparently the most important group of these
enzymes. Lysosomal cathepsins can be categorized as cys-
teine (cathepsins B, C, F, H, K, L, O, S, V, W, and X), aspartic
(cathepsins D and E) and serine (cathepsin G) proteases
(75, 125, 245). They have their pH optima *5, although sev-
eral of them remain active in a neutral milieu, in a timeframe
varying from minutes (cathepsin L) to hours (cathepsin S)
Lysosomes fuse with autophagosomes=endosomes to form
‘‘hybrid’’ organelles containing material in the course of
degradation that originates both from the outside and inside
of the cell. After completed degradation of the enclosed ma-
terial, lysosomes turn into ‘‘resting’’ organelles, which in the
electron microscope look homogeneous and moderately
electron dense. They are then ready for new rounds of fusion.
The pronounced fusion and fission activity that is such a
typical characteristic of the lysosomal compartment (146) al-
lows lytic enzymes and other lysosomal contents to be dis-
tributed between different lysosomes (Fig. 7).
Because of autophagy of iron-containing macromolecules,
such as ferritin and mitochondrial complexes, the lysosomal
compartment is rich in iron (Fig. 8) that partly exists in redox-
active form, making lysosomes sensitive to oxidative stress
through intralysosomal Fenton-type reactions (this section).
The hydroxyl radicals that form may give rise to peroxidation
of material under degradation, resulting in lipofuscin forma-
tion (this section) or, if substantial, in lysosomal membrane
permeabilization (LMP) (Fig. 9). Lysosomal destabilization,
with relocalization to the cytosol of potent hydrolytic en-
zymes andlow-mass iron, is able toinduce either apoptosis or
necrosis. As is schematically shown in Fig. 10, cross-talking
between lysosomes and mitochondria is an important process
in apoptosis, and either of these organelles may induce the
process. Obviously, a concerted and balanced action of lyso-
somal cathepsins andcytosolic caspasesis requiredfor typical
apoptosis, whereas a domination of cathepsins will give rise
to necrosis (129).
After receptor-mediated endocytosis, the initially plasma
membrane–bound receptors are often, but not always, re-
turned to the plasma membrane, whereas the ligands are
mostly propagated down the lysosomal compartment. One
exception to this ‘‘rule of ligand degradation’’ is transferrin,
which is returned to the plasma membrane together with its
receptor, whereas the iron which is bound to transferrin is
released into the late endosomes because of their acidic en-
vironment (pH*6) and transported to the cytosol by trans-
port proteins such as Nramp [reviewed in (129)].
The processing and presentation of antigens in immuno-
competent cells is dependent on a form of endocytotic–
exocytotic activity, whereas autophagic degradation is vital
not only for the normal turnover of cellular constituents, but
using the sensitive cytochemical sulfide silver method.
Glutaraldehyde-fixed specimens are exposed to ammonium
sulfide at pH*12 and then developed in a colloid-protected
(gum arabic) solution containing silver lactate and the re-
ducing agent hydroquinone. Tiny silver particles precipitate
and gradually enlarge to a size visible by light microscopy.
The process is akin to physical development of a photo-
graphic plate. After a short development time of 25min (A),
only very iron-rich lysosomes are visible (arrows). These ly-
sosomes most probably correspond to autophagolysosomes
that are engaged in the degradation of iron-containing ma-
terial, such as ferrritin or mitochondrial complexes. After
40min of development (B), a strong general lysosomal pat-
tern is seen, reflecting the fact that most lysosomes contain
some low-mass iron.
Demonstration of lysosomal iron in HeLa cells by
516 TERMAN ET AL.
also for the elimination of damaged structures and cytosolic
microorganisms that have invaded the cell. Some cell types
are able to exocytose lysosomal contents or even intact lyso-
somes (secretory lysosomes) [reviewed in (146)]. It has been
recognized that tumor cells often secrete lysosomal proteases,
which, in combination with the previously described acidifi-
cation of their surroundings that enhances the activity of ly-
sosomal proteolytic enzymes, may help them to infiltrate and
metastasize (58, 144, 151, 165, 181, 189, 249). The acidic mi-
croenvironment around highly malignant and rapidly grow-
ing tumor cells may be used in tumor therapy. Drugs (for
example, weak acids) that are deprotonated in the neutral
environment of normal cells, and consequently charged, are
less likely to pass the plasma membrane of normal cells.
cross-talk. Lysosomes are involved in
the external as well as in the internal
apoptotic pathway. In the external
pathway, lysosomal destabilization can
be mediated by caspase 8, either di-
rectly or indirectly, through activation
of Bax or by ceramide that is converted
into sphingosine, which is a lysosomo-
tropic detergent. P53 can also destabi-
lize lysosomes through the recently
discovered LAPF protein. A variety of
synthetic lysosomotropic agents (e.g.,
MSDH or 3-aminopropanal) can labi-
lize lysosomes. Furthermore, the lyso-
somal membrane can be peroxidized
and subsequently ruptured by hy-
droxyl radicals that originate from
peroxide and intralysosomal redox-
can further damage lysosomes either
directly or through activation of phos-
pholipases. The internal apoptotic pathway is activated through mitochondrial damage. This could be the result of activation
of Bax or Bid, phospholipases, or lysosomal enzymes with subsequent cytochrome c release and the start of the caspase
cascade, leading to apoptosis.
hydroxyl radicals. Hydrogen
peroxide is formed normally,
mainly from mitochondria. It
is efficiently inactivated by the
Only a small portion of this
lysosomes, a compartment rich
in cystein and redox-active
from the degradation of a va-
riety of iron-containing pro-
teins. Hydrogen peroxide and
iron react in the Fenton reac-
tion, yielding hydroxyl radi-
cals. This process gives rise
to intralysosomal oxidation=
undergoing autophagic degra-
dation. Some oxidation prod-
ucts polymerize and become
undegradable (lipofuscin) and
accumulate in lysosomes of
which do not dilute the pig-
ment by division.
9. Resultsof in-
MITOCHONDRIAL TURNOVER AND AGING517
Conversely, the same drug would remain protonated and
uncharged in the acidic microenvironment around malignant
cells and thus be able to pass through their plasma mem-
branes unhindered. An example of such compounds that
succinate (a-TOS). The principle behind the enhanced uptake
of this compound and other weak acids by tumor cells that
acidify their surroundings can easily be demonstrated by
exposing cells in culture to a-TOS at a different pH of the
medium. At a slightly acidic pH of the medium (*6), cells are
much more affected than they are at neutral pH (170).
As pointed out earlier, lysosomes fuse with autophago-
somes, or deliver part of their content (‘‘kiss-and-run’’), to
form autophagolysosomes (Fig. 7). Here a variety of organ-
elles and proteins are degraded into their building blocks,
which in turn are reused by the anabolic machinery of the cell
after their transport to the cytosol [reviewed in (122, 241)].
From a physiological point of view, the lysosomal compart-
ment can be looked upon as a box, built of vacuoles that
constantly fuse and divide, that receives enzymes from the
cell. After substrate degradation inside individual lysosomes,
the products diffuse or are actively transported to the cytosol
Because many iron-containing macromolecules are de-
graded intralysosomally, low-mass iron is released inside the
lysosomal compartment. Because the lysosomes also contain
reducing agents (for example, glutathione), ascorbic acid, and
the amino acid cysteine, some low-mass iron exists as Fe(II)
with the capacity to generate highly reactive radicals if ex-
lysosomes are very sensitive to oxidative stress, and their
membranes easily peroxidized and permeabilized by the
radicals that are formed secondary to the Fenton-type reac-
tions taking place in the lysosomes. The rupture of lysosomes
in apoptosis or necrosis, depending on the magnitude of this
This is because some of them have recently been active in
autophagic degradation, whereas others have not. A thresh-
old for LMP seems to exist after oxidative stress, probably
because of the high concentration of vitamin E within lyso-
somal membranes (253). It also is important to remember
that LMPafteroxidativestress isnotaninstantaneous process
but rather requires some time, because peroxidation and the
ensuing fragmentation of lysosomal membranes require
Keeping the concentration of redox-active iron in lyso-
somes as low as possible is consequently important for the
mass iron from lysosomes to the cytosol is thus important, as
well as ways of temporarily binding iron in a non–redox-
active form (129).
Autophagy is a nonstop biologic renewal mechanism pro-
vidinglysosomal degradationofthe cell’sownconstituents. It
represents one of the main pathways for the turnover and
reutilization of worn-out long-lived proteins and organelles
and is a perfectly normal process. Interestingly, the multi-
catalytic proteinase complexes, proteasomes, which also play
an important role in the turnover of macromolecules, are
themselves degraded by autophagy (53). The implication of
this is that hampered autophagy might result in defective
proteasomes, because they, in common with mitochondria
and other organelles, are then not properly renewed. The
mechanisms involved in the formation of the autophagic
double membrane (the phagophore), the inclusion of materi-
als to be degraded, and the fusion of autophagosomes and
lysosomes were mostly recently worked out as a result of
the discovery in yeast of a large family of phylogenetically
well-preserved autophagy-related genes (ATG) (122, 207,
To date, three different mechanisms of autophagy have
been described in mammalian cells: macroautophagy (also
known as just autophagy), microautophagy, and chaperone-
mediated autophagy (CMA). Macroautophagy, which, in at
least a subset of cases is a nonselective process (202), involves
the sequestration within a double membrane–bounded vac-
uole of portions of the cytosol, including aggresomes, dys-
functional mitochondria or proteasomes, as well as long-lived
soluble proteins. The initially formed sequestration vacuole is
devoid of lysosomal enzymes and is termed an autophago-
some. In consecutive steps, it fuses with lysosomes and with
other sequestration vacuoles, eventually resulting in the for-
mation of an autophagolysosome (also called autolysosome),
amino acids and other monomeric molecules occurs (49, 129,
265); see Fig. 7.
Macroautophagy is the most universal type of autophagy,
being involved in the degradation of practically any type of
such as starvation, to generate ATP and essential building
blocks by means of the nonspecific degradation of organelles
and cytosolic macromolecules that are not critical for the
survival of the cell (233).
Microautophagy is probably also involved in the turnover
of lysosomes themselves, as suggested by the fact that fibro-
blasts exposed to the sequestration inhibitor 3-methyladenine
accumulate large numbers of altered lysosomes containing a
lipofuscin-like material (222). In support of this view, lyso-
somes with active hydrolytic enzymes have been found
within autophagosomes (162). Moreover, by immunoelectron
demonstrated to exist inside lysosomes and not only in the
surrounding membranes (11). Such membrane fragments are
particularly abundant within lysosomes of I-cell disease fi-
broblasts that lack active acid hydrolases (198).
Microautophagy involves the invagination of portions of
the lysosomal membrane into the lumen of the lysosome=
vacuole, resulting in the internalization of cytosolic com-
partments. A variation on this process, known as ‘‘piecemeal
microautophagy of the nucleus’’ or PMN, has also been re-
cently described in yeast, in which a small nonessential sec-
tion of the nucleus is ‘‘pinched off’’ at nucleus–vacuole (NV)
junctions (for a review, see ref. 130). Microautophagy is also a
possible means of lysosomal membrane turnover, apparently
working in parallel with macroautophagy.
CMA is a selective mechanism of lysosomal degradation,
specific for soluble cytosolic proteins, which contain a tar-
geting motif biochemically related to the penta-peptide
KFERQ (for Lys-Phe-Glu-Arg-Gln). Unlike other forms of
518 TERMAN ET AL.
mammalian autophagy, CMA does not require vesicle for-
mation or major changes in the lysosomal membrane to pro-
ceed: rather, substrate proteins directly cross the lysosomal
membrane to reach the lumen, where they are rapidly de-
graded. This pathway requires the cooperation of cyto-
solic and lysosomal chaperones, including cytosolic hsc70
(cyt-hsc70), lysosomal hsc70 (lys-hsc70), LAMP-2A, and co-
chaperones including Hsp90, Hsp40, Hip, Hop, and Bag-1 (1).
The upregulation of CMA is observed in response to nutri-
tional starvation and mild stress induced by toxic compounds
or oxidants (49, 265).
Subsequent to cellular damage, reparative autophagy fol-
lows, by which altered and malfunctioning structures are re-
placed. Such reparative autophagy is commonly seen, for
example, after ionizing irradiation, virus infection, and hyp-
period of starvation is overbridged by a period of enhanced
autophagy in the liver, explaining why certain mutations that
hinder autophagy are lethal (262). Recent evidence suggests
that regular day-long periods of starvation may, by stimu-
lating autophagy, help to ‘‘keep cells clean’’ and be beneficial
C. Autophagic degradation
of mitochondria (mitophagy)
Autophagy is the most efficient mitochondrial turnover
mechanism, providing for the complete removal of irrevers-
ibly damaged mitochondria (mitophagy). It is believed that
mitochondria are normally replaced every 2–4 weeks in rat
brain, heart, liver, and kidneys (161), although recent studies
have shown that the turnover rate might be considerably
higher (163). Mitophagy is particularly important for long-
lived postmitotic cells, whose mitochondria have pronounced
oxidative damage (see Section II.C). Under stable, normal
environmental conditions, the biogenesis of mitochondria
through mitochondrial fission is balanced by mitophagy, re-
sulting in a relatively constant number of mitochondria
within postmitotic cells. As a result of fission, oxidatively
damaged mitochondrial biomolecules are diluted, as are also
damaged components of dividing cells. Mitophagy, in turn,
prevents an excessive accumulation of mitochondria. The
regulatory link between mitochondrial fission and mitophagy
follows from a recent study showing that the pro-fission mi-
and the formation of autophagic vacuoles that contain mito-
As mentioned earlier, oxidant-induced injury to mito-
chondrial components initiates asymmetric mitochondrial
and, probably, with different degrees of oxidative damage)
(12, 247); see Fig. 4. Conceivably, asymmetric mitochondrial
fission would provide for the selective removal by mitophagy
of damaged mitochondria with abnormal membrane poten-
tials, butwhetherthishypothesisiscorrectremains tobe seen.
Mitophagy is apparently less critical for the survival of
constantly proliferating cells, in which mitochondria formed
during fission are distributed between daughter cells, pre-
venting the excessive accumulation of damaged mitochon-
dria. It was found that, in cultured pancreatic b-cells, the
frequency of mitochondrial fission events is 5 to 10 times
higher than the frequency of mitophagy (40, 45). The hy-
pothesis that mitophagy is of particular importance for post-
mitotic cells is strengthened by our observation that the
exposure of cultured rat cardiac myocytes to an inhibitor of
autophagic sequestration, 3-methyladenine, induces exces-
sive accumulation of defective mitochondria, followed by
degeneration and death (237).
The adaptive (reparative) role of mitophagy increases un-
der stress conditions, in particular with the oxidative stress
associated with increased mitochondrial damage (142). Mi-
tophagy is also an essential component of the programmed
cell death (PCD-2, or autophagic cell death) that develops in
the presence of excessive cellular damage (44, 118).
Questions yet unanswered are whether mitophagy is se-
lective and how mitochondria are targeted for degradation.
Early studies performed on isolated hepatocytes exposed to
amino acid starvation showed that macroautophagy is a
nonselective process (202), suggesting that the degradation of
mitochondria occurs randomly. This is probably true for
starving cells, in which autophagy activation is a rescue
mechanism, using the cells’ own constituents to generate en-
ergy and limited vital anabolism. All the functions of starving
cells, perhaps with the exception of autophagy, are down-
regulated, with many mitochondria, including normal ones,
However, increasing evidence suggests that both under
normal and some pathologic conditions, mitochondria are
selectively removed by autophagy. For example, the autop-
hagic removal of mitochondria has been found essential for
the maturation of yolk sack–derived embryonic erythroid
cells (229). Furthermore, it has been shown that the mito-
chondria of degenerating embryonic flight muscles, as well as
sperm mitochondria undergoing degradation after oocyte
are tagged by ubiquitin (55, 208, 226). It remains unclear what
types of damage result in mitochondrial ubiquitination, and
to what extent ubiquitin is required for mitophagy.
Recently, a number of new protein molecules involved in
mitochondrial autophagy were discovered. The mitochon-
drial outer membrane protein, Uth1p (mitochondrial outer
membrane and cell wall–localized SUN family member re-
quired for mitochondrial autophagy), was found essential
for mitophagy in yeast (121). In addition, it was shown
that a yeast mitochondrial intermembrane-space protein
phosphatase homologue, Aup1p, is required for efficient
stationary-phase mitophagy and cell survival (230). Several
mitophagy-specific proteins have been demonstrated to exist
in mammalian cells. A Bcl-2 family member protein, Bnip3,
seems to trigger mitochondrial autophagy in mouse embryo
fibroblasts that are exposed to hypoxia (203) and in neonatal
rat cardiac myocytes during ischemia=reperfusion-like injury
been found to play an essential role in mitophagy associated
with erythroid maturation in mice (197). The relation between
these mitophagy-related proteins and ubiquitination remains
Although the molecular mechanisms responsible for se-
lective mitochondrial degradation remain to be further
elucidated, extensive evidence indicates that it is oxidant-
induced mitochondrial damage and related mitochondrial
permeability transition with decreased inner-membrane po-
tential that often initiate a sequence of events resulting in
MITOCHONDRIAL TURNOVER AND AGING519
mitophagy (reviewed in ref. 119, 246). As noted earlier, this
kind of mitochondrial damage is an important characteristic
of aging and a variety of pathologies.
VI. Lipofuscin Formation and Its Influence
A. Influence of labile iron and ROS
on lipofuscin formation
Lipofuscin (age pigment) is a nondegradable, yellowish-
brown, autofluorescent, polymeric compound that slowly ac-
correlated with species longevity (76, 123) and reviewed in refs.
29 and 232. This interesting fact in and of itself suggests that
lipofuscin accumulation may be hazardous to cells.
As can be seen from the previous sections, the aging of a
multicellular organism depends largely on alterations occur-
ring in long-lived postmitotic cells, such as neurons, cardiac
myocytes, and RPE cells, whereas replicative aging due to
preventing malignant transformation (206). Long-lived post-
mitotic cells are very rarely (or never) replaced through the
division and differentiation of stem cells, thus allowing lipo-
fuscin and other biologic waste materials (such as irreversibly
damaged mitochondria and aberrant proteins) to accumulate
decay and cell death (241). It should be noted that lipofuscin
also accumulates in cultured cells undergoing replicative se-
nescence (associated with a progressive decline in the cellular
proliferation rate), or in cells whose proliferation is inhibited
by pronounced density-dependent inhibition of growth (re-
form in various cell types, but only long-lived nondividing
cells accumulate significant amounts of the pigment.
It is now generally accepted that the aging of long-lived
postmitotic cells is at least partly induced by endogenously
formed ROS, affecting various cellular structures, but mainly
mitochondria and lysosomes (10, 98, 124, 128, 132, 212, 239).
Lipofuscin formation is one of the most important manifes-
tations of ROS-induced damage that occurs within the lyso-
somal compartment (27).
Although rapid and effective, lysosomal (autophagic) deg-
radation is not completely perfect. Even under normal
conditions, some iron-catalyzed peroxidation occurs intra-
lysosomally (as pointed out, lysosomes are rich in redox-
active iron), resulting in oxidative modification of the
autophagocytosed material, making it resistant to the hy-
drolytic activity of lysosomal enzymes. If cells do not divide,
this material progressively accumulates in the form of lipo-
fuscin inclusions. Lysosomes receive a wide variety of au-
tophagocytosed subcellular structures, most importantly
mitochondria, which are rich in lipidaceous membrane com-
ponents and iron-containing proteins, such as cytochrome c.
That lipofuscin=ceroid to a large extent originates from mi-
synthase subunit c in age pigment or ceroid granules (72). In
Alzheimer disease (AD), large amounts of mitochondrial li-
poic acid have been found associated with lipofuscin, which
indicates pronounced mitochondrial autophagy (166). Thus
mitochondria not only are the main generators of ROS, trig-
gering lipofuscinogenesis, but also are a major source of the
macromolecules from which lipofuscin forms.
Lipofuscinogenesis has pronounced similarities to the for-
mation of advanced glycation end products (AGEs) that is
mainly involved in the aging of connective tissue components
leading to wrinkling of the skin, cataract, the stiffening of
blood vessels, etc. (see earlier). These effects are thought to be
a consequence of alterations in extracellular matrix proteins,
which undergo polymerization and lose their elasticity be-
cause of glycosylation with accompanying Maillard reactions
(the binding of carbonyls of reducing sugars to amino groups
of proteins) and ensuing Amadori reorganization (the for-
mation of new carbonyls within the complex, thus allowing
proteins to stiff, plastic-like structures) (60). The difference
between formation of AGEs and lipofuscin is that no sugars
are involved in the formation of the latter, because then the
linking aldehydes are produced by degradation of oxidized
lipids. Basically, lipofuscin may be regarded as a nonde-
gradable plastic-like polymer that slowly matures by in-
tramolecular reorganization. It has been found that lipofuscin
contains oxidized proteins in which tyrosine residues have
been replaced by DOPA (3,4-dihydroxy-l-phenylalanine, an
oxidized form of tyrosine) (67, 117, 191, 201), secondary to an
oxidation that most probably is mediated by redox-active
iron. Because DOPA, being a hydroquinone-type structure, is
capableofredoxcycling(190),itisconceivable that lipofuscin,
because of its oxidized protein residues, produces superoxide
and hydrogen peroxide. This production might result in the
labilization of the surrounding membrane, especially because
lipofuscin is also rich in loosely bound iron. Consequently,
lipofuscin-loaded lysosomes may be sites of pronounced
Fenton-type reactions and be especially sensitive to oxidative
stress (26, 29, 110).
Oxidized proteins are usually considered degraded by the
proteasome system secondary to ubiquitination, but recent
studies have shown that such proteins may also undergo
autophagic degradation. Studies using a cell line with a
thermolabile ubiquitin-conjugating enzyme showed that ox-
idized proteins were still being degraded at a normal rate,
even in the absence of functioning ubiquitin-conjugating en-
zymes (209). Although it is not clear to what extent other
pathways for ubiquitin conjugation could have been active,
the latter finding suggests that the proteasomal pathway is
not the only one involved in the degradation of oxidized
By using an approach in which oxidatively modified pro-
teins were generated in vitro by allowing cells to incorporate
DOPA into proteins, it was demonstrated that mildly modi-
fied proteins were efficiently degraded by proteasomes, be-
cause this process could be inhibited by the specific
proteasome inhibitor, lactacystin (192). By increasing the
amount ofDOPA incorporated into proteins, it was, however,
possible to generate proteins that were heavily modified and
eventually to generate lysosomal autofluorescent lipofuscin
aggregates in cells (68). Moreover, by using inhibitors of the
proteasome and lysosomal proteases, it was found that the
degradation of the more highly modified aggregate-prone
DOPA-containing proteins began to switch from the protea-
somal to the lysosomal pathway (192). This ‘‘cooperation
between proteasomes and lysosomes’’ may suggest that
substantially modified proteins are no longer substrates
for proteasomes and, therefore, must be redirected to the
520TERMAN ET AL.
and ubiquitinated misfolded proteins localize to proteasome-
rich perinuclear sites, whereas terminally aggregated proteins
are sequestered in autophagic vacuoles (111). Further evi-
dence for cross-talk between the proteasomes and lysosomes
was reported for human RPE cells, in which an accumulation
of perinuclear ubiquitin-, Hsp70-, and LAMP2-positive ag-
gregates occurred in response to MG-132 (a proteasome in-
hibitor), with the aggregates being removed after cessation of
inhibition by a mechanism thought to involve autophagy
(196). In an earlier study, increased levels of lipofuscin-like
autofluorescence were found in cultured SH-SY5Y neuro-
blastoma cells exposed to the proteasome inhibitor MG-115
(224). It is to be noted that the accumulation of lipofuscin-like
material and other protein aggregates, considered an effect of
proteasome inhibition, may depend also on the inhibition of
lysosomal functions. MG-115 and MG-132, which belong to
the peptide aldehyde proteasome inhibitors, are known to
suppress not only proteasomes but also lysosomal cathepsins
(39, 160). Even stronger support for proteasomal–lysosomal
crosstalk was provided by a study using a highly specific
proteasome inhibitor, MG-262. When exposed to this sub-
stance, cultured human fibroblasts showed increased lipo-
fuscin accumulation (especially pronounced under hyperoxic
conditions), suggesting that modified proteins that were not
degraded by proteasomes underwent autophagic degrada-
tion, contributing to lipofuscinogenesis (242).
In professional scavengers, such as RPE cells and macro-
phages (foam cells) in atheroma, a large portion of lipofuscin
(or ceroid) originates from endocytosed material (135, 171).
Dependingon the nature
endocytosed material, the composition of lipofuscin varies
among different types of postmitotic cells, and no chemical
formula can be given for this complex substance that seems to
of the autophagocytosed=
be composed mainly of cross-linked protein and lipid resi-
dues. It should be pointed out, however, that all forms of
lipofuscin contain considerable amounts of redox-active iron,
which, as was pointed out in the Introduction, sensitizes
Figure 11 shows the basic mechanisms behind lipofuscino-
B. Consequences of the nondegradability of lipofuscin
The accumulation of lipofuscin within the lysosomal com-
partment apparently compromises autophagic degradative
capacity, prolonging the half-lives of long-lived proteins and
organelles and creating a situation in which cells are forced
Consistent with this theory, the capacity for autophagic deg-
radation is found to be diminished in aged lipofuscin-loaded
cells (52, 231, 236), which may lead to some serious conse-
quences. For example, the delayed degradation of mitochon-
dria would result in increased damage by self-produced ROS,
additionally contributing to lipofuscinogenesis and perhaps
inducing apoptotic cell death by LMP.
Recently, an elegant study on Caenorhabditis elegans added
new evidence for the hypothesis that lipofuscin accumulation
is causally related to aging and the deterioration of post-
mitotic cells. Fortunately, these tiny nematodes are transpar-
ent, which permits lipofuscin to be measured directly with
spectrofluorimetry in vivo. It was found that mutant nema-
todes that live either longer or shorter than the wild-type
animal accumulate lipofuscin at a slower or quicker pace,
respectively. It also was found that calorie-restricted worms
lived longer and accumulated lipofuscin more slowly than
did animals fed ad libitum. Finally, when wild-type siblings
pofuscin formation. Super-
mitochondria as a side prod-
uct of biologic respiration. It
is converted into hydrogen
peroxide (H2O2) by superox-
ide dismutase (SOD). Hy-
drogen peroxide is further
homolytically split, yielding
the hydroxyl radical (HO?), in
the presence of ferrous iron
(the Fenton reaction). Hy-
droxyl radicals damage sur-
throughout the cell. Oxida-
tively damaged macromole-
cules (parts of mitochondria
and other cellular structures)
enter lysosomes through au-
tophagy. In the autophago-
lysosomes, which are rich in
iron, more hydroxyl radicals
damage to autophagocytosed material, resulting in its polymerization and undegradability (i.e., lipofuscin formation). Ac-
tions of lysosomal enzymes (Enz) and reactive oxygen species are indicated as dashed arrows. Black dots, oxidatively
damaged macromolecules, including components of lipofuscin. Bold curved arrows, the sequence of events.
Mechanisms of li-
??) forms mainly in
MITOCHONDRIAL TURNOVER AND AGING 521
that ageddifferently, as evaluated by changes in their motility
capacity, were compared, it was found that the still mobile
and youthful ones at day 11 of their life spans contained only
25% of the lipofuscin that was found in severely motility-
impaired siblings of the same age. This implies that lipofuscin
accumulation reflects biologic rather than chronologic
Besides accumulating the intralysosomal ‘‘waste’’ material
lipofuscin, aging postmitotic cells also form extralysosomal
‘‘garbage,’’ such as damaged dysfunctional mitochondria and
indigestible protein aggregates (aggresomes) that for some
reason are not efficiently autophagocytosed or degraded by
the proteasome pathway (reviewed in refs. 233 and 235).
Aged mitochondria are enlarged and show considerably re-
duced fusion and fission activity. Their autophagy may be
prevented by their size, because the autophagy of large
structures is apparently energy consuming, and autophago-
somes seem to have an upper volume limit (169, 241). These
most important phenomena are discussed in detail in Section
VII of this review.
The accumulation of aberrant proteins within aging post-
mitotic cells is a consequence of both ROS-induced damage
and the incomplete degradation of altered protein molecules.
Although damaged proteins may partially preserve their
functions, their enzymatic activity per unit mass declines
(186, 216). Aberrant proteins often show a tendency to aggre-
gate. Lewy bodies and neurofibrillary tangles (composed of
a-synuclein or the hyperphosphorylated protein tau, respec-
tively) are characteristic examples of such aggregates (17, 96).
The importance of autophagy for the removal of protein
aggregates and delaying aging was demonstrated in a recent
study on Drosophila (210). During normal aging of the fly, the
expression of the Atg8a gene (which is of importance for au-
tophagy) decreases in neurons, resulting in the accumulation
of aggregates of ubiquitinated proteins. When the expression
of Atg8a was upregulated, the aggregates disappeared, and
the flies showed an increased resistance to oxidative stress, as
well as a more than 50% prolonged life span.
C. Disease-related accumulation of intralysosomal
and extralysosomal waste
When, as a result of damage, proteins and other macro-
molecules undergo modifications that make them indigest-
ible, or when lysosomal degradation is suppressed, or
autophagy increases greatly as a result of reparative efforts
(reparative autophagy), the accumulation of intralysosomal
waste may occur more rapidly than in normal aging. The
trans-Golgi network (TGN) and by secretory vesicles transported to late endosomes that acidify and maturate into lysosomes
(see Fig. 7), which in turn fuse with autophagosomes (APSs). The continual fusion and fission of the lysosomal vacuoles
ensures the distribution of acid hydrolases within the lysosomal compartment, including APS. In contrast to a young cell (A)
that has only few lysosomes containing the undegradable age-pigment lipofuscin (Lf), senescent postmitotic cells (B) contain
large numbers of Lf-containing lysosomes, to which more and more lysosomal enzymes are directed in a useless effort to
degrade lipofuscin. These lysosomal enzymes are lost for useful purposes (e.g., for the degradation of newly autophagocy-
tosed material), resulting in a delayed turnover and the accumulation of waste products. Damaged=dysfunctional mito-
chondria are indicated by dark shading.
The accumulation of ‘‘waste’’ is a consequence of imperfect autophagy. Lysosomal enzymes are produced in the
522 TERMAN ET AL.
amount of nondegradable material can increase, for example,
as a result of enhanced damage to cellular structures due to
various types of stress, ionizing irradiation, intoxications, or
malnutrition (85). Lysosomal functions can also be sup-
pressed as a result of the administration of drugs, such as the
lysosomotropic agent chloroquine that increases the lyso-
somal pH and thereby depresses the activity of lysosomal
hydrolytic enzymes, or in a large variety of lysosomal-storage
diseases that are associated with genetic defects of specific
lysosomal hydrolases (256). Intralysosomal waste material
formed under these conditions is usually called ‘‘ceroid’’ or
‘‘ceroid-type lipofuscin.’’ Ceroid also forms because of in-
creased sensitivity to oxidation, such as in vitamin E defi-
ciency (78). This material has, however, practically the same
physicochemical properties (i.e., natural yellow-brown color,
autofluorescence, electron density after osmium fixation, re-
sistance to hydrolysis) and basic mechanisms of formation
(oxidant-induced, aldehyde-mediated intra- and intermolec-
ular cross-linking) as true, age-related, lipofuscin.
Because of the variable composition of lysosomal pigment
inaginganddifferentdiseases, aswellasindifferenttissues, a
distinction between lipofuscin and ceroid can reasonably be
made only from an etiologic viewpoint, but not with respect
to their properties and chemistry (29). As ceroid forms more
quickly than lipofuscin, the latter is perhaps just a more-
restructured and advanced polymer (compare its formation
with the Amadori reorganization of glycosylated proteins).
Juvenile neuronal lipofuscinosis is associated with lyso-
somal lipofuscin=ceroid accumulation and is a nontypical
example of a lysosomal storage disease in the sense that no
enzyme deficiency has been demonstrated. This fatal disease
rather seems to be caused by a mutation of a lysosomal
membrane protein of importance for the fusion between ly-
sosomes and autophagosomes. As a result, the influx of de-
grading enzymes to autophagosomes is slow (35), allowing
more time for peroxidation and polymerization of lysosomal
contents into lipofuscin=ceroid. In cell cultures exposed to
lysosomal protease inhibitors, this scenario is reproduced,
also an effect of the exposure of cells in culture to oxidative
stress [e.g., growing cells at 40% ambient oxygen (reviewed in
refs. 29 and 232)].
In the progressive eye condition known as age-related
macular degeneration (AMD), the accumulation of lipofuscin
within retinal pigment epithelial (RPE) cells in the macular
area of the retina is a warning sign of developing disease (77).
build-up of deposits in the Bruch membrane (an inflamma-
tory process with prominent genetic involvement) and in
choroidal neovascularization. Exactly how LF accumulation
inside RPE cells results in their degeneration and the eventual
death of the RPE cells themselves, as well as the photorecep-
tors that they are supporting, is not understood in detail.
Findings from RPE cells in culture, however, have provided
some information: LF acts as a sensitizer to blue light by
causing singlet oxygen formation with ensuing membrane
destabilization, the release of hydrolytic enzymes to the cy-
tosol, and resulting apoptosis. Moreover, lysosomal LF ac-
cumulation prevents normal RPE phagocytic activity (258).
This is probably a result of a misdirection of newly produced
lysosomal enzymes to lipofuscin-loaded lysosomes at the
expense of late endosomes and autophagosomes (Fig. 12).
To better understand the molecular mechanisms behind
AMD, and, hopefully, to improve our ability to interfere with
this common disease, it is necessary to understand how RPE
cells for years are able to handle the extremely high demands
on their capacity for lysosomal degradation without being
overwhelmed by LF accumulation. It has been suggested that
iron overload may exacerbate AMD: (a) postmortem AMD-
affected eyes showed an excess of both chelatable and non-
chelatable iron in the RPE cells and in the Bruch membrane,
including drusen; (b) the iron-carrier protein transferrin is
upregulated at both the mRNA and protein levels in patients
with AMD compared with controls (43); and (c) mice with
RPE iron overload resulting from a deficiency of the iron ex-
porters ceruloplasmin and hephaestin develop retinal de-
generation with some features of AMD, including sub-RPE
deposits and subretinal neovascularization. These findings
point to a dysfunctional regulation of lysosomal labile iron in
AMD. The same may be true for other neurologic disorders in
which abnormal accumulation of lipofuscin occurs in the ly-
Extralysosomal deposition of oxidized proteins is reported
in age-related pathologies, including neurodegenerative dis-
orders such as AD (225) and Parkinson diseases (PD) (191),
atherosclerosis and cataractogenesis (59), and diabetic com-
plications (15). Interestingly, the increased levels of oxidized
proteins that are normally observable in tissues from older
animals are less prominent after CR, suggesting that semi-
starvation may lead to a reduction in the extent of protein
Multiple neurodegenerative disorders, including AD, PD,
and Huntington disease (HD), are characterized by selective
neuronal loss concomitant with the accumulation of in-
traneuronal proteinaceous inclusionbodies. These include the
amyloid plaques and neurofibrillary tangles consisting of the
hyperphosphorylated protein tau in AD, Lewy bodies con-
sisting of a-synuclein in PD, and nuclear inclusions in the
polyglutamine-repeat diseases, such as HD. Intracellular in-
clusions are thought to form when the proteasome capacity is
the presence of mutated a-synuclein (153). When misfolded
proteins accumulate in sufficient quantity, they are prone to
aggregation. Pathologic inclusions have been shown to con-
tain the core protein as well as ubiquitin (Ub) moieties,
chaperones and components of the ubiquitin-proteasome
system (UPS), thus implicating the UPS in disease progres-
The accumulation of a-synuclein, Ub, and other proteins in
Lewy bodies contained within degenerating dopaminergic
neurons in idiopathic PD suggests that the inhibition of
normal=abnormal protein degradation may contribute to
neuronal death (154). In support of this, McNaught and co-
workers (154) reported a reduction in all three catalytic ac-
tivities of the 20=26S proteasome in the substantia nigra in PD
by 39%, 42%, and 33% for chymotryptic, tryptic, and caspase-
like activity, respectively. Direct proteasome inhibition in
cultured rat ventral mesencephalic cells and PC12cells also
led to the formation of a-synuclein=ubiquitin-containing in-
tracytoplasmic inclusion bodies and the preferential degen-
eration of dopaminergic neurons, further implicating UPS
dysfunction in PD (155). Support for the dysfunctional UPS
hypothesis is provided by rare hereditary forms of PD,
in which mutations in the genes encoding parkin (an E3
MITOCHONDRIAL TURNOVER AND AGING523
Ub-ligase) and ubiquitin c-terminal hydrolase L1 or UCH-L1
(a deubiquitinylating enzyme), both components of the UPS,
are closely linked to disease progression.
VII. Imperfect Mitochondrial Turnover and Postmitotic
A. Age-related accumulation of defective mitochondria
within postmitotic cells
The number of defective (senescent) mitochondria within
long-lived postmitotic cells progressively increases with age.
These mitochondria show structural deterioration, such as
swelling, lossof cristae, or destruction of the inner membrane,
often combined with mitochondrial enlargement, leading
to the formation of the so-called giant mitochondria (Figs. 2
and 3). These structural changes underlie mitochondrial
functional deficiencies such as decreased ATP production
[reviewed in (240)].
Senescent mitochondria appear in initially young and
healthy postmitotic cells, where they slowly accumulate with
time. This suggests that early mitochondrial changes are sto-
chastic by nature and perhaps reflect insufficient mtDNA re-
Because occasional senescent mitochondria are found early
in the life span of postmitotic cells, the malfunction of mito-
chondrial renewal mechanisms is obviously not a conse-
quence of aging but rather an inherent characteristic. Another
example of the inherent insufficiency of cellular homeostasis
is the formation of oxidatively modified, undegradable ma-
terial (lipofuscin) within lysosomes (29, 30). Even if the initial
changes in mitochondria and lysosomes obviously are not
caused by aging, they accumulate as a function of time and,
moreover, trigger additional (secondary) pathogenic mecha-
nisms responsible for mitochondrial and lysosomal aging.
According to the extensive experimental evidence, these
mechanisms are associated either with an age-related de-
crease in the degradation of defective mitochondria and their
components, or with the increased replication of damaged
organelles. The involvement of additional pathogenic mech-
anisms, which are described later, suggests that the course of
aging accelerates with time.
B. Age-related decline in autophagy and Lon protease
activity accelerates mitochondrial damage
Decreased lysosomal degradative capacity and, in partic-
of increased mitochondrial damage with age. Autophagy has
been shown to decline in aged rat and mouse hepatocytes (63,
231). In a recent study, an age-related decrease in autophagic
degradation was found for rat hepatocyte mitochondria
containing oxidatively damaged mtDNA (as assessed by the
increased 8OHdG levels). When autophagy was stimulated
by the antilipolytic agent 3,5-dimethylpyrazole, the content of
8OHdG decreased to the levels comparable to those in young
animals (37). The loading of lysosomes with lipofuscin is
of autophagy in aged cells (see also Section VI). As predicted
by the mitochondrial–lysosomal axis theory of aging (30),
lipofuscin-loaded lysosomes would consume a major part of
the newly produced lysosomal hydrolases that, however,
cannot digest the undegradable material. As a result, a lesser
amount of lysosomal enzymes remains available for autop-
hagic degradation, including mitophagy. Consequently,
damaged mitochondria accumulate, producing increased
levels of ROS and leading to further lipofuscin accumulation.
Senescent mitochondria and lipofuscin-loaded lysosomes
gradually replace normal organelles, finally resulting in cell
death due to a lack of ATP. In agreement with this theory,
heavily lipofuscin-loaded growth-arrested human fibroblasts
showed significantly decreased autophagy, and they poorly
survived amino acid starvation (236). Furthermore, in aged
neonatal rat cardiac myocytes, the amount of lipofuscin pos-
itively correlates with mitochondrial damage and ROS for-
Lon protease expression has also been shown to be affected
by oxidative stress and aging, leading to the accumulation of
carbonylated mitochondrial proteins in mouse skeletal mus-
cles (23). Because Lon proteases are coded for by nuclear
genes, their downregulation may take place independent of
any initial mitochondrial damage.
C. Enlarged mitochondria are resistant to degradation
and do not fuse with normal ones
A number of the mechanisms involved in the progress of
mitochondrial alterations with age are triggered by primary
changes in the mitochondria themselves. One such change
may be mitochondrial enlargement. Because autophagy is an
energy-dependent process, the degradation of large organ-
elles is obviously more demanding than that of small ones.
The initial enlargement of some mitochondria is quite pre-
dictable, for example, because of the oxidative damage to
mitochondrial membranes and proteins that can disturb fis-
sion. Indeed, the aging of cultured Chang cells, which were
kept at low oxidative stress, was associated with the accu-
mulation of enlarged mitochondria and was paralleled by the
downregulation of Fis1 (264). Furthermore, siRNA depletion
of enlarged and flattened mitochondria with decreased
membrane potential and elevated ROS production, resulting
in DNA damage and increased b-galactosidase activity (139).
The latter is most probably not an age-specific process, but
rather reflects the generally enhanced production of lyso-
somal enzymes and reduced cell growth (205).
Larger mitochondria would have a lesser chance to be au-
tophagocytosed and would thus undergo progressive de-
generation and enlargement, resulting in the gradual
appearance of giant mitochondria; this, along with lipofuscin
accumulation within postmitotic cells, is an age-related, irre-
versible phenomenon. That mitochondrial size does matter
for the propensity of mitochondria to be autophagocytosed
follows from the fact that the inhibition of autophagy with
3-methyladenine in neonatal rat cardiac myocytes results in
a dramatic accumulation of small-sized mitochondria with
low membrane potential and only a moderate increase in the
number of large (giant) senescent-like mitochondria (237); see
Figs. 2 and 3. The most likely interpretation of these results
is that mitochondria that are excluded from recycling by
3-methyladenine administration accumulate in quantities re-
flecting their normal turnover rates. The gradual accumula-
tion of large mitochondria that occurs during normal aging
resembles, to some extent, a bottleneck phenomenon: only
524 TERMAN ET AL.
mitochondria that are small enough are autophagocytosed
[i.e., passthe bottleneck (233)];see Fig. 13.Consistent withour
results, the accumulation of small depolarized mitochondria
was observed in INS1cells exposed to 3-methyladenine, as
well as in autophagy-deficient Beclin 1 RNAi H4cells and
ATG5?=?mouse embryonic fibroblasts (246).
The irreversibility of an age-related mitochondrial enlarge-
ment also follows from our recent observation that the pop-
ulation of senescent-like giant mitochondria formed within
cultured rat myoblast cells exposed to 3-methyladenine does
not fuse and exchange their contents with normal mito-
chondria (see Figs. 6, 14, and 15) A decreased mitochon-
drial inner-membrane potential and downregulation of
Mfn2 and OPA1 are the most apparent reasons for the
inability of giant mitochondria to fuse (169). Thus, giant
mitochondria seem progressively to amass with age because
they do not fuse or divide or they are removed by macro-
That cells cannot rid themselves of giant mitochondria is
consistent with earlier observations of the impaired fusion
capacity of damaged mitochondria with increased OPA1
degradation (see Section III.B).
cumulation of giant mitochondria. Autophagy of
large mitochondria is more complicated than that of
small ones. This results in progressive accumula-
tion within long-lived postmitotic cells of enlarged
(giant) mitochondria, which do not ‘‘pass the bottle-
neck.’’ (A, B) Young and senescent cells, respec-
tively. Inhibition of autophagic sequestration with
3-methyladenine (C), which suppresses the turnover
of all mitochondria independent of their size, results
in the accumulation of mitochondria in quantities
reflecting their turnover rates. Consequently, cells
accumulate numerous small mitochondria and only
few large, senescent-like mitochondria.
The bottleneck model of age-related ac-
mitochondria in L6 rat myoblast cells. (A)
Fluorescence-microscopy image of a myoblast
cell containing normal mitochondria (up to
0.5mm wide). (B) Several mitochondria are dra-
matically enlarged. The highlighted mitochon-
drion is 4.5mm long and 2.6mm wide. (C) The
width distributions of 80 normal (light bars) and
80 giant (dark bars) mitochondria as determined
by analysis of fluorescent images in SimplePCI
5.3 software. The distributions were normalized
by dividing the number of hits in each bin (bin
size, 0.2mm) by 80 (i.e., the total number of mi-
tochondria analyzed). Reprinted from Navratil
et al. (169) with permission from Elsevier.
Morphology of normal and giant
MITOCHONDRIAL TURNOVER AND AGING525
D. Mechanisms of the age-related accumulation
of mitochondria with homoplasmic mtDNA mutations
It has been found that aging postmitotic cells often accu-
mulate homoplasmic mtDNA point mutations and deletions,
resulting in the gradual replacement of all normal mito-
chondria with mutated ones (116, 176). Mitochondria with
homoplasmic mutations have also been found to occupy
atrophic segments of the ragged muscle fibers typical of aged
mtDNA was earlier described for a variety of pathologies,
mainly myopathies and cardiomyopathies (reviewed in refs.
62 and 149). It was hypothesized that the accumulation of
mitochondria with single-type mtDNA mutations within
postmitotic cells is because mutated mtDNA is associated
with defective respiration, resulting in decreased oxidative
damage to mitochondrial membranes. As a result, such mi-
tochondria would be less targeted by autophagy than normal
ones (56). This theory, called SOS (for ‘‘survival of the slow-
est’’) requires, however, some confirmation that mitochondria
are indeed being targeted for autophagy based on the degree
of damage to their membranes. Moreover, the accumulating
evidence that mitochondria with decreased membrane po-
tential are preferentially selected for degradation (see earlier)
is contradictory to the SOS hypothesis.
Another still unproven explanation for the accumulation of
mutated mitochondria within aged or diseased postmitotic
cells is that mutant mtDNA has a replicative advantage over
normal mtDNA, resulting in the clonal expansion of mito-
chondria with mutated DNA (176). Although it has been
suggested that deleted mtDNA might replicate more easily
mutations cannot be explained from this premise (57). Yet, an
important argument in favor of the clonal expansion hy-
pothesis is the accumulation within malignant tumor cells of
mitochondria with homoplasmic mtDNA mutations (103).
These cells and their mitochondria replicate indefinitely,
which excludes the preferential accumulation of mitochon-
dria with single-type mutations because of their limited au-
tophagy, as was predicted by the SOS hypothesis that
describes the age-related accumulation of defective mito-
chondria specifically in long-lived postmitotic cells.
E. Decreased mitochondrial biogenesis in aged cells
Age-related decrease of mitochondrial biogenesis (143) is
related, at least in part, to diminished AMPK activity (185,
188). AMPK appears to be the key cellular energy sensor,
linking decreased mitochondriogenesis to several aging-
associated changes, including insulin resistance and deficient
lipid metabolism (185, 188). With aging, a decline in PGC-1a
expression levels also occurs, with the latter being slowed by
CR (8, 100). Treatment with hexarelin or resveratrol rescues
mitochondrial biogenesis and lipid metabolism by inducing a
higher turnover of aged and damaged organelles (194). It
are tissue specific, being more dramatic in the central nervous
system (143), which is consistent with the generally higher
susceptibility of neurons and other postmitotic cells to the
The relation between proteins involved in the regulation of
mitochondrial dynamics and age-related mitochondrial
changes is not understood. An important fact in this regard is
that the processing of the fusion protein OPA1 may be regu-
immunostained for cytochrome c oxidase subunit I (COXI, green) and mitochondrial fusion proteins OPA1 and Mfn2 (red).
The nuclei are counterstained with DAPI in the composite images. Relative OPA1 and Mfn2 expression levels were assessed
by measuring the red fluorescence signal normalized by the green COXI fluorescence in overlapping areas. Bottom plots
show differences in the distributions of Opa1=COXI (left) and Mfn2=COXI (right) ratios between normal (blue) and giant
(red) mitochondria. For each distribution a median value (Med) is reported. Modified from Navratil et al. (169) with
permission from Elsevier. (For interpretation of the references to color in this figure legend, the reader is referred to the web
version of this article at www.liebertonline.com=ars).
Expression of mitochondrial fusion proteins in giant mitochondria. L6 myoblasts treated with 5mM 3-MA were
526 TERMAN ET AL.
lated by cellular ATP levels (9), thereby providing a connec-
tion with aging- or disease-associated bioenergetic decline. It
was found that depolarization of mitochondria is followed by
the proteolyticcleavageofOPA1long(l)-isoforms (69,90,105,
mitochondrial fragmentation (69, 246). The dependence of
OPA1 proteolysis on ATP concentration may explain how
even small changes in the membrane potential can change
mitochondrial morphology (101). Mitochondrial energetics
regulates OPA1 levels through the presenilin-associated
AFG3L2-proteases (46, 70, 105). The short form of OPA1 has
been detected in cells with dysfunctional respiration and in
patients with cardiomyopathies. It would be interesting to
levels in the aging tissue because of the ATP deficiency of
senescent cells. This regulatory mechanism would result in
mitochondrial fragmentation and autophagic turnover of
mitochondria. However, the ability of postmitotic cells to re-
new their mitochondria would progressively decline because
of decreasing autophagic capacity and the increasing pro-
portion of enlarged senescent mitochondria that can neither
fuse nor divide (169).
Possible mechanisms involved in age-related mitochon-
drial damage are summarized in Table 3.
VIII. Summary and Conclusions
Mitochondria are the power-generating factories ofthe cell,
but they are also generators of ROS. These reactive species are
probably responsible for at least a major part of the macro-
molecular damage that gradually accumulates and results in
mitochondrial malfunction. Mitochondrial failure deprives
the cell of ATP and increases the production of ROS, which
eventually leads to cell death. Age-related mitochondrial
damage mainly affects postmitotic cells, such as neurons and
myocardial cells, which are rarely or not at all replaced be-
cause of the division or differentiation of stem cells. Mi-
tochondria possess a potent proteolytic self-repair system, as
well as a capacity to fuse and divide. During fusion, mito-
chondria can probably renew themselves by exchanging
material with other mitochondria, whereas mitochondrial di-
vision enhances the dilution of damaged macromolecular
components, which apparently prevents the aging of actively
proliferating cells. In addition, mitochondria can divide
asymmetrically into two daughter organelles, one of which
containsmost ofthe defects. This allowsa selective mitophagy
damaged beyond the possibility of effective repair. If this
system worked perfectly, the cell would remain vital and op-
erating well. In postmitotic cells, however, over time, a slow
accumulation of poorly functioning mitochondria occurs;
these oftentake the phenotype ofgiant mitochondriawith low
membrane potential. The reason that these dysfunctional mi-
tochondria accumulate seems to be an increasing incapability
to divide, in combination with insufficient autophagic degra-
dation. The regulation of mitochondrial fusion and fission is
just beginning to be at least partly understood, whereas the
influence of accumulated lipofuscin on the process of macro-
autophagy, as a result of extensive studies of C. elegans and
cells in culture during the last few years (50, 74, 122, 129), is
somewhat better understood. The new findings point to the
existence of a mitochondrial–lysosomal cross-talk in which
formation of ROS by mitochondria gives rise to peroxidation
of autophagocytosed lysosomal contents under degradation.
compartment of long-lived postmitotic cells; this is catalyzed
by the redox-active iron that is released during the degrada-
tion of autophagocytosed ferruginous materials. Lipofuscin
can neither be degraded nor exocytosed, to any substantial
extent. Lipofuscin accumulation, in turn, seems to depress the
capacity of lysosomes to degrade autophagocytosed materials
because of their futile endeavor to break down the plastic-like,
undegradable pigment. Moreover, the transport and loca-
tion of an increasing amount of newly produced lysosomal
enzymes to lipofuscin-loaded lysosomes seems to create a
Table 3. Potential Mechanisms Involved in Age-related Mitochondrial Changes due to Oxidative Damage
Mechanism Cellular manifestations
1. Decreased capacity of cellular-degradation mechanisms
A. Decreased mitophagy due to lipofuscin loading of
A. Accumulation of defective, mainly enlarged
mitochondria combined with extensive lipofuscin
loading of lysosomes
B. Intramitochondrial accumulation of oxidatively
B. Decreased activity of mitochondrial proteases
2. Decreased susceptibility of mitochondria and their
components to degradation
A. Mitochondrial enlargement (presumably due
to impaired fission) interferes with mitophagy
B. Modifications of mitochondrial proteins make them
resistant to Lon, Clp-like, and AAA proteases
C. Defective respiration may lessen oxidative damage
to mitochondrial membranes, resulting in decreased
targeting of mitochondria for autophagy
3. Increased replication (clonal expansion) of defective
4. Decreased mitochondrial biogenesis
A. Accumulation of enlarged (giant), functionally
B. Intramitochondrial accumulation of oxidatively
C. Accumulation of mitochondria with reduced respiration,
presumably with homoplasmic mtDNA mutations
3. Accumulation of mitochondria with homoplasmic
4. Lack of normal mitochondria
Detailed explanations and references are given in the text.
MITOCHONDRIAL TURNOVER AND AGING527
an insufficient amount of degrading capacity, thereby forcing
the cell to use damaged structures, including mitochondria,
longer than is optimal. It is envisioned that further research
focused on mitochondrial–lysosomal interactions will add to
our knowledge about aging and age-related pathologies, as
well as suggest new strategies for antiaging intervention.
We thank Carol Makkyla for proofreading the manuscript.
E.A.A. thanks NIH R01-AG20866 for support.
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Address correspondence to:
Department of Clinical Pathology and Cytology
Karolinska University Hospital
141 86 Stockholm
Date of first submission to ARS Central, March 21, 2009; date
of final revised submission, July 22, 2009; date of acceptance,
August 2, 2009.
AGE¼advanced glycation end product
AMD¼age-related macular degeneration
Aup1p¼yeast mitochondrial protein phosphatase
Bnip3L¼Bcl2=adenovirus E1B 19-kDa interacting
Drp1¼dynamin-related protein 1
Fis1¼mitochondrial fission protein 1
Fzo¼fuzzy onion protein
Hsp70¼heat-shock protein 70
LAMP2¼lysosome-associated membrane protein 2
LMP¼lysosomal membrane permeabilization
Miro¼mitochondrial rho protein
Miro-2¼mitochondrial rho-2 protein
MTP18¼mitochondrial protein 18
mtTFA¼mitochondrial transcription factor A
Nramp¼natural resistance–associated macrophage
NRF1¼nuclear respiratory factor 1
OPA1¼optic atrophy protein 1
PCD¼programmed cell death
receptor g, coactivator 1 a
RIP140¼nuclear receptor–interacting protein
ROS¼reactive oxygen species
RPE¼retinal pigment epithelium
SIRT1¼sirtuin (silent mating-type information
regulation 2 homologue) 1 (C. cerevisiae)
SNARE¼soluble N-ethylmaleimide-sensitive factor
SOS hypothesis¼‘‘survival of the slowest’’ hypothesis
Uth1p¼mitochondrial outer membrane and cell
wall–localized SUN family member
required for mitochondrial autophagy
MITOCHONDRIAL TURNOVER AND AGING 535