Experimental Gerontology 35 (2000) 119 –131
Review
When lysosomes get old夞
Ana Maria Cuervo, J. Fred Dice
Department of Physiology, Tufts University School of Medicine, Boston, MA, USA
Received, 27 September, 1999; received in revised form, 23 December, 1999; accepted, 23 December, 1999
Abstract
Changes in the lysosomes of senescent tissues and organisms are common and have been used
as biomarkers of aging. Lysosomes are responsible for the degradation of many macromolecules,
including proteins. At least five different pathways for the delivery of substrate proteins to
lysosomes are known. Three of these pathways decline with age, and the molecular explanations for
these deficiencies are currently being studied. Other aspects of lysosomal proteolysis increase or do
not change with age in spite of marked changes in lysosomal morphology and biochemistry.
Age-related changes in certain lysosomal pathways of proteolysis remain to be studied. This area of
research is important because abnormalities in lysosomal protein degradation pathways may contribute to several characteristics and pathologies associated with aging. © 2000 Elsevier Science
Inc. All rights reserved.
Keywords: Aging; Senescence; Protein degradation; Lipofuscin deposits; ␤-amyloid deposits; Lysosomal;
Endosomal system
1. Introduction
Since first being described by DeDuve in the 1960s as “lytic bodies,” lysosomes have
been considered to be a likely site of degradation of proteins and other macromolecules
(Bowers, 1998). We use the name lysosomes to refer to a degradative compartment
surrounded by a single membrane and containing hydrolases that operate optimally at
acidic pH (Dice, 2000). Endosomes are vesicles that form at the plasma membrane and
contain materials that will eventually be delivered to lysosomes. We recognize that
endosomes may also contain some hydrolases so that the distinction between endosomes
and lysosomes may blur in certain cells.
Recent research has identified five different pathways by which lysosomes can take up
intracellular and extracellular proteins (Cuervo and Dice, 1998; Dice, 2000). Age-related
夞This work was supported by the National Institute of Aging AG008290 (A.M.C.) and AG06116 (J.F.D.).
* Corresponding author. Tel.: ⫹617-636-6707; fax: ⫹617-636-0445.
E-mail address: jdice@opal.tufts.edu (J. Dice)
0531-5565/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved.
PII: S 0 5 3 1 - 5 5 6 5 ( 0 0 ) 0 0 0 7 5 - 9
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A. M. Cuervo, J. F. Dice / Experimental Gerontology 35 (2000) 119 –131
Table 1.
Effect of age on total protein degradation in different organs in rats
Organ
Age
(young-old, months)
Protein degradation
(% decrease with age)
Whole body
Liver
Heart
1–20
6–22
1.5–12
12–24
0.8–26
56.1 ⫾ 3.1
50.0 ⫾ 4.1
28.3 ⫾ 0.9
46
62.5 ⫾ 2.2
4
4
4
1
2
0.8–25
5–25
0.8–25
0.8–24
12–24
64.4 ⫾ 12.2
⫺53.7
0
44.3
88.2 ⫾ 0.7
11
1
3
1
2
Skeletal muscle
Red
White
Lung
Kidney
Brain
Studies
(number)
Data presented are modified from (Ward & Shibatani, 1994). Values are the medium ⫾ SD of the changes in
total protein breakdown in different rat tissues described in studies from different groups. Original experiments
used different methods to measure protein degradation rates. All values shown correspond to experiments
measuring total protein degradation. Assays analyzing only cytosolic proteins or specific organelle proteins were
not included.
decreases in some of these proteolytic pathways have been documented, whereas other
pathways increase or do not change with age. Still other lysosomal proteolytic pathways
have not yet been studied with regard to aging. Several characteristics of aged cells and
organisms may result from the reduced lysosomal protein degradation pathways, including
the increased cellular protein content of senescent cells and the accumulation of proteins
with inappropriate posttranslational modifications (Dice, 1993).
2. Reduced protein degradation rates in aging
In most tissues of aged organisms and in most aging model systems, including
nematodes, fruit flies, and cultured fibroblasts, overall proteolysis declines with age (Table
1). This decline is postponed in rats and mice by caloric restriction in proportion to the
degree of life span extension (Ward and Shibatani, 1994). Caloric restriction alters the
expression of many different genes, so the explanation for how it maintains protein
degradation rates may be complex (Martin et al., 1996). The reduced proteolysis with
aging is most evident for long-lived proteins, some of which are known to be substrates
for lysosomal pathways of proteolysis. Important nonlysosomal proteolytic pathways
include the ubiquitin-proteasome pathway (DeMartino and Slaughter, 1999) and the
calpains (Carafoli and Molinari, 1998). The ubiquitin–proteasome pathway shows only
minor changes with age (Shibatani and Ward, 1996) except in certain tissues like the lens
and under specific circumstances such as oxidative stress (Shang et al., 1997). Most
studies of age-related changes in calpain activities conclude that they increase rather than
decrease (Glaser et al., 1994; Saito et al., 1993). These considerations caused a focus on
lysosomal protein degradation pathways as the most likely explanation of reduced protein
degradation in aging.
Many initial studies of intracellular protein degradation in aging were performed in
nematodes (Prasanna and Lane, 1979; Reznick and Gershon, 1979), but such studies were
A. M. Cuervo, J. F. Dice / Experimental Gerontology 35 (2000) 119 –131
121
Fig. 1. Lysosomal pathways of protein degradation. Different proteolytic pathways share the lysosome as the
final compartment for the degradation of their substrate proteins. Plasma membrane proteins and some extracellular proteins are degraded after endocytosis. Secretory proteins located in secretory vesicles reach the
lysosomal matrix by crinophagy. Three different types of autophagy (macroautophagy, microautophagy and
chaperone-mediated autophagy) contribute to the degradation of cytosolic proteins and proteins located inside
other organelles. See the text for description of these pathways. L, lysosomes; ER, endoplasmic reticulum; M,
mitochondrion; AV, autophagic vacuole; SV, secretory vesicle; G, Golgi; N, nucleus; PM, plasma membrane.
limited to whole-body protein turnover. More recent studies that used rodent tissues or
cultured human fibroblasts have shown that alterations in lysosomal proteolytic pathways
apply to many different aging model systems. In addition, the recent availability of
transgenic mice for different age-related pathologies (e.g. Alzheimer’s disease, neurodegenerative disorders, systemic amyloidosis, etc.) has become very helpful for identifying
the role of the lysosomal system in the pathogenesis of those diseases.
3. Lysosomal pathways of protein degradation
Lysosomes are able to take up and degrade both extracellular and intracellular proteins
(Fig. 1). Endocytosis in its various forms can internalize extracellular proteins as well as
intracellular membrane proteins. Secretory proteins can be delivered to lysosomes by
fusion of the secretory vesicle membrane with the lysosomal membrane rather than with
the plasma membrane. This process has been called crinophagy. Cytoplasmic proteins can
be taken up by lysosomes by microautophagy, macroautophagy, or chaperone-mediated
autophagy. The age-related changes in lysosomal function described below are summarized in Table 2.
3.1. Endocytosis
Internalized extracellular and plasma membrane proteins are commonly delivered to
lysosomes for degradation (Fig. 1). However, many examples exist in which plasma
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A. M. Cuervo, J. F. Dice / Experimental Gerontology 35 (2000) 119 –131
Table 2.
Age-related changes in the lysosomal pathways of protein degradation
Lysosomal degradation pathway
Change with age
Endocytosis
Fluid phase
Absorptive
Receptor mediated
Crinophagy
Macroautophagy
Chaperone-mediated autophagy
Microautophagy
⫽
⫽
⫽, 2
1
2
2
?
Original references in (Cuervo & Dice, 1998; Dice, 2000).
membrane proteins are, instead, recycled to the plasma membrane. The molecular signals
within proteins that lead to their delivery to lysosomes or to their recycling to the cell
surface are currently being defined (Dice, 2000).
Fluid-phase endocytosis and absorptive endocytosis are not affected by age when
normalized to cellular protein content (Gurley and Dice, 1988). Early studies reported no
changes in receptor-mediated endocytosis (Lee et al., 1982), but more recent results with
a wide variety of substrates for receptor-mediated endocytosis have uncovered age-related
decreases in activity (Vetvicka et al., 1985). The mechanisms responsible for these
impairments vary depending on the protein in question and the cell type analyzed. For
example, decreased receptor number, decreased ligand binding, decreased receptor internalization, and defective receptor recycling to the plasma membrane have all been
reported. The causes of these age-related defects may be a reduction in receptor protein
synthesis, oxidative damage to the receptor, and/or an altered cytoskeletal organization
(Dini et al., 1996; Malorni et al., 1998). It is especially important to determine whether or
not receptor-mediated endocytosis of proteins modified by advanced glycosylation end
products is decreased in aging, as reduced catabolism of advanced glycosylation end
product-modified proteins in the circulation might contribute to their accumulation in old
age (Araki et al., 1992).
3.2. Crinophagy
Lysosomes are able to degrade secretory proteins sorted through either constitutive or
regulated secretory pathways (Dice, 2000). The most common form of crinophagy
involves direct fusion between secretory vesicles and lysosomes (Fig. 1). Other forms of
this process include macroautophagic engulfment of secretory vesicles (see below) and
selective transfer of secretory materials to regions of the endoplasmic reticulum that then
receive lysosomal enzymes to form a lysosome that is already full of secretory proteins.
These processes seem mechanistically quite distinct and should be given different names
once more is known about them. The proportion of a secreted protein that is degraded by
crinophagy is regulated. For example, when insulin secretion is needed because of high
blood glucose, less is degraded by crinophagy and more is secreted. Insulin secretion is
decreased by aging, and part of this decrease is due to higher rates of crinophagy perhaps
caused by a defect in the ␤ cells’ ability to detect blood glucose (Borg et al., 1994).
Increased crinophagy in other endocrine and immune cells might similarly contribute to
reduced secretion of polypeptide hormones and cytokines (Bi et al., 1998).
A. M. Cuervo, J. F. Dice / Experimental Gerontology 35 (2000) 119 –131
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3.3. Macroautophagy
Areas of cytoplasm, including complete organelles such as mitochondria or peroxisomes, can be sequestered by a double-membraned organelle called an autophagic vacuole
(Fig. 1). These vacuoles first lose their outer membrane by unknown mechanisms, then
acidify due to a proton pump in their inner membrane, and next fuse with lysosomes.
Macroautophagy can operate in a largely nonselective fashion (Seglen et al., 1990), but
also with selectivity for certain organelles over others (Yokota, 1993). Genetic analyses of
macroautophagy in yeast have uncovered a novel protein conjugation cascade similar to
ubiquitination that is required for formation of autophagic vacuoles (Mizushima et al.,
1998). Macroautophagy is activated in liver and other tissues of intact animals by short
periods of starvation. Glucagon enhances whereas insulin and certain amino acids suppress macroautophagy (Mortimore et al., 1996).
Macroautophagy decreases with aging because of a decreased formation of autophagic
vacuoles combined with an even more striking delay of fusion of autophagic vacuoles
with lysosomes (Terman, 1995). These changes result in an accumulation of autophagic
vacuoles in tissues from old animals even though the flux of proteins through these
structures is diminished. Quantification of the age-related decrease in macroautophagy
was possible by comparing the half-lives of proteins microinjected into the cytosol of
young and senescent human fibroblasts in which macroautophagy had been activated
because of their confluence, but chaperone-mediated autophagy (see below) was suppressed by the addition of serum growth factors to the medium (Dice, 1982). The
degradation rates of such proteins declined 3-fold in senescent cells.
3.4. Chaperone-mediated autophagy
There is also a selective pathway of autophagy that is responsible for the degradation
of certain cytosolic proteins after their direct transport through the lysosomal membrane
(Fig. 1; Cuervo and Dice, 1998; Dice, 2000). All of the substrate proteins contain a
peptide-targeting sequence related to the pentapeptide, KFERQ. This motif is recognized
by a molecular chaperone, the heat shock protein of 73kDa (hsc73) and co-chaperones
(Fig. 2, step 1). The substrate and chaperones then bind to a receptor in the lysosomal
membrane identified as the lysosomal-associated membrane protein type 2a (lamp2a; Fig.
2, step 2). Lamp2a belongs to a family of lysosomal membrane proteins with similar
structural characteristics. The larger part of the protein is located in the lysosomal lumen,
and they contain a single transmembrane region and a short cytosolic tail. Substrate
proteins bind to the cytosolic region of lamp2a (Cuervo and Dice, 1996). After binding to
lamp2a, the substrate is then transported into the matrix (Fig. 2, step 3). Transport of
substrate proteins requires the presence of another chaperone, lysosomal hsc73 (ly-hsc73),
in the lysosomal matrix. In human fibroblasts in culture, the ly-hsc73 is required for the
import of substrate proteins (Agarraberes et al., 1997). Rat liver lysosomes also require
ly-hsc73 for the operation of chaperone-mediated autophagy (Cuervo et al., 1997). Once
in the lysosomal matrix, the substrate proteins are rapidly degraded by lysosomal proteases (Fig. 2, step 4).
This pathway of proteolysis is activated in animal tissues in response to prolonged
starvation. Macroautophagy is activated by shorter periods of starvation, but then it
decreases back to basal levels. In cultured cells that are confluent, macroautophagy has
already been activated, and serum deprivation activates chaperone-mediated autophagy.
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Fig. 2. Hypothetical model of chaperone-mediated autophagy. 1, Substrate proteins interact with hsc73 and
cochaperones (hsc73/cochap) in the cytosol; 2, the complex is directed to the lysosomal surface where it binds
to lamp2a; 3, the substrate protein is transported into the matrix assisted by the lysosomal-hsc73 (lys-hsc73); 4,
the substrate is completely degraded by the cathepsins (cath).
The activity of this pathway is regulated most closely by the amount of lamp2a in the
lysosomal membrane. To achieve maximal activity during long periods of starvation,
ly-hsc73 levels also increase. The levels of lamp2a at the lysosomal membrane are tightly
regulated by two different mechanisms. Its degradation rate is reduced under conditons
that activate chaperone-mediated autophagy. Lamp2a is degraded within lysosomes, and
this degradation is initiated by two different proteases in the lysosomal membrane (Cuervo
and Dice, submitted). The truncated form of lamp2a is thus released from the membrane
into the matrix, where it is rapidly degraded. In addition, there is also a significant portion
of intact lamp2a in the lysosomal lumen (Jadot et al., 1996). The intact matrix lamp2a
originates from deinsertion of lamp2a from the lysosomal membrane (Cuervo and Dice,
submitted). In addition, part of the lamp2a in the matrix can be reinserted back into the
lysosomal membrane (Cuervo and Dice, submitted) as has also been shown for other
membrane proteins (Economou and Wickner, 1994), and this reinsertion contributes to the
increase in lamp2a levels at the lysosomal membrane when chaperone-mediated autophagy is activated.
A reduction in chaperone-mediated autophagy in senescent fibroblasts was shown by
microinjection of substrate proteins and measurement of their degradation in the absence
of serum to activate this particular proteolytic pathway (Dice, 1982). This reduced activity
is also evident using in vitro assays with isolated lysosomes from senescent fibroblasts or
from livers of aged rats. In both cases, the defective activity with age seems to be at least
in part caused by reduced levels of lamp2a in the lysosomal membrane (Cuervo and Dice,
submitted). No alterations were found in the levels or activities of hsc73, and levels of
A. M. Cuervo, J. F. Dice / Experimental Gerontology 35 (2000) 119 –131
125
ly-hsc73 actually increased in lysosomes from old animals, perhaps as an attempt to
compensate for the reduced lamp2a levels.
3.5. Microautophagy
Lysosomes can sequester cytoplasmic components in invaginations or finger-like
protrusions of their membranes and, after a membrane fusion event, the material resides
within an intralysosomal vesicle (Fig. 1). The vesicle membrane first disappears, then the
internalized material is digested (Dice, 2000). Microautophagy is thought to account for
the lysosomal component of degradation of cytosolic proteins in mammalian cells under
optimal growth conditions. However, microautophagy also occurs in yeast, where it is
responsible for the enhanced degradation of peroxisomes under certain conditions in
which these organelles are no longer needed (Subramani, 1998). Distinct steps in the
process have been identified (Sakai et al., 1998), and genetic analysis shows that the same
protein conjugation cascade required for macroautophagy is also required for microautophagy. Nothing is known about age-related changes in microautophagy
4. Causes of reduced lysosomal proteolysis with age
An increased volume and fragility of lysosomes are common findings for tissues from
senescent organisms, but there is no evidence for overt lysosomal rupture in aged cells.
Compared to other intracellular membranes, lysosomal membranes are very sensitive to
free radical damage (Hochshild, 1971), and an age-related increase in oxidation of lipids
or proteins within the lysosomal membrane may be the cause of the increased fragility,
reduced fusion of lysosomes with autophagic vacuoles, and/or reduced levels of lamp2a
in the lysosomal membrane. On the other hand, the reduced levels of lamp2a in the
lysosomal membrane from old cells may result from anything causing an increase in its
degradation rate or a decrease in its synthetic rate. Further experiments are required to
resolve these issues.
Lysosomes from senescent organisms are filled with the aging pigment, lipofuscin,
along with other indigestible materials (Terman and Brunk, 1998). The most likely source
of indigestible materials is from cross-linking between macromolecules. For example, free
oxygen radicals can cross-link proteins through isopeptide bonds, and such bonds are not
readily cleaved by lysosomal cathepsins. Such cross-linking may take place before the
protein enters lysosomes, but it may also occur within the lysosomal matrix (Peterson et
al., 1998). There is no direct evidence that lipofuscin is harmful to the cells, but it has been
reported to interfere with autophagic vacuole formation (Terman et al., 1999). It is also
possible that lipofuscin in the lysosomal matrix somehow alters trafficking of proteins to
the lysosomal membrane. The finding that fluid phase and absorptive endocytosis are
unaltered by aging and that crinophagy is elevated suggests that lysosomal function is not
uniformly compromised.
There are a variety of age-associated changes in lysosomal hydrolase activities, but the
alterations seem to vary depending on the hydrolase assayed and the tissue used (Cuervo
and Dice, 1998). In none of these cases has the altered hydrolase activities been shown to
cause abnormal lysosomal function. In general, activities of lysosomal proteases are not
rate-limiting in the degradation of proteins. Rather, components of the pathways of
delivery of substrates to the lysosomal matrix usually limit protein degradation rates
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(Dice, 1999). Therefore, the assorted changes in lysosomal hydrolase activities reported
during aging may not be significant. More relevant could be any age-related change in the
intralysosomal pH or in the levels of cystatins, the physiological inhibitors inside lysosomes, that will modify the activity of most cathepsins. Unfortunately, no detailed studies
on these topics have yet been published.
In addition to age-related changes in lysosomes, changes in the substrate proteins also
need to be considered as contributors to reduced proteolysis in aging. Most of the
protein-covalent modifications common in aging such as oxidation, glycation, phosphorylation, deamidation, carbonyl modification, and failure to correctly fold (Gafni, 1997),
result in changes in their proteolytic susceptibility (Sukharev et al., 1997). Furthermore,
modification of amino acids may destroy or generate peptide sequences required for entry
into a particular proteolytic pathway (Gracy et al., 1998).
The cooperative interaction between the different intracellular proteolytic systems in
normal conditions might also lead to some of the age-related changes in the lysosomal
activities. For example, in some degenerative conditions, some of the ubiquitinated
proteins are degraded by nonlysosomal and/or lysosomal systems depending on the stage
of degeneration of the structures (Li and Greenwald, 1997). Also, a decrease in the activity
of intraorganelle proteases (e.g. in mitochondria) could result in the formation of undigested proteolytic intermediates that then might accumulate within secondary lysosomes
after autophagy of the whole organelle (Lee and Wei, 1997).
5. Effects of reduced lysosomal proteolysis with age
As mentioned earlier, reduced degradation rates of proteins contributes to their accumulation of a variety of posttranslational modifications. Such abnormal proteins may
induce stress-responsive genes (Choppra et al., 1997). Reduced degradation can also
account for an age-related increase in the protein content of cells. When this increased cell
size is not observed in models of aging, there must be an equivalent decline in rates of
protein synthesis (Makrides, 1983). Reduced macroautophagy and chaperone-mediated
autophagy may also limit the senescent cells’ ability to mobilize amino acids under
starvation conditions. A reduced rate of protein degradation also causes proteins to be
slower to reach new steady-state levels after a change in their synthetic rate. Therefore,
reduced rates of proteolysis will contribute to the slower adaptability to changes in the
environment noted for senescent cells and organisms (Jurivich et al., 1997).
Reduced lysosomal proteolytic pathways may also contribute to age-associated pathologies. Poor handling of modified proteins by aged cells have been proposed to happen in
age-related pathologies such as diabetes, atherosclerosis and neurodegenerative diseases
(Dean et al., 1997). The ␤-amyloid precursor protein (APP), a type-I transmembrane
protein expressed in most mammalian cells, is abnormally processed by what is called an
amyloidogenic pathway in Alzheimer’s Disease (AD). The fragments resulting from that
processing (AP) accumulate in amyloid deposits typical of this disease. In the early-onset
form of AD, mutations in presenilin 1 and presenilin 2, secretases located in the endoplasmic reticulum and early Golgi apparaturs, are the most common cause of the disease
(Haas and Mandelkow, 1999). For the late-onset form of AD, which accounts for more
than 90% of AD, an age-related decline in the degradation of AP seems to be mainly
responsible for the formation of abnormal aggregates (Chu et al., 1998). Under normal
conditions part of the AP secreted by neurons is directly degraded by extracellular
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127
proteases but some is also reinternalized by endocytosis and undergoes degradation in the
endosomal/lysosomal system of neurons and glial cells (Chu et al., 1998). Lysosomes are
able to degrade the Alzheimer’s precursor protein into nonamiloidogenic peptides and
amino acids (Paresce et al., 1996). A reduced lysosomal proteolytic activity in AD may
allow the intralysosomal aggregation of the amyloidogenic peptides and its further
excretion to form the amyloid deposits (Chu et al., 1998; Frautschy et al., 1998).
In other degenerative diseases, such as the neuronal ceroid lipofuscinosis or Batten
disease, a specific mutation in the lysosomal tripeptidyl-peptidase I that completely
abolishes its peptidase activity has been identified in patients with a late onset form of the
disease (Sleat et al., 1997; Vines and Warburton, 1999). The major protein that accumulates in the lysosomes of those patients is subunit c of mitochondrial ATP synthase, an
extremely hydrophobic protein (Kominami et al., 1992). The peptidase activity must be
required for efficient lysosomal degradation of this protein in particular (Tomkinson,
1999).
Abnormal activation of particular lysosomal pathways of proteolysis in specific senescent tissues and organs might lead to pathologic states such as reduced hormone secretion
(Bednarski et al., 1997). The reduction of insulin secretion with aging has already been
noted to be caused, at least in part, by an increased fraction of insulin being diverted for
lysosomal degradation by crinophagy (Borg et al., 1994). However, other defects in the
lysosomal proteolytic pathways in some other endocrine tissues have also been reported.
Thus, the reduced thyroid hormone formation with aging is mainly due to reduced
endocytosis and lysosomal degradation of the precursor of these hormones, thyroglobulin
(van den Hove et al., 1998). Changes in endocytosis and/or crinophagy may similarly lead
to reduced levels of cytokine receptors or circulating cytokine levels and thus contribute
to the decrease in the immune response typical of old organisms (Miller, 1999). Reduced
lysosomal protein degradation can also affect the generation of antigenic peptides for
presentation on major histocompatability complex (MHC) class II and possibly MHC
class I pathways (Vetvicka et al., 1985).
Finally, though not properly a lysosomal proteolytic pathway, the consequences of
age-related changes in the normal release of cathepsins to the extracellular medium need
to be considered. Those released cathepsins, in conjunction with the matrix metalloproteases, play an important role in maintenance and remodeling of the extracellular matrix
(Werb, 1997). A decreased activity of matrix proteases, including released cathepsins, in
most connective tissues is responsible for the impaired wound healing in senescent
organisms (Werb, 1997) and also has been implicated in atherosclerosis (He et al., 1996)
6. Altered lysosomal proteolysis and causes of aging
Several fundamental causes of aging recently have been proposed. For example,
progressive shortening of telomeres is associated with senescence, and maintenance of
telomere length slows aging in cultured fibroblasts (Wright et al., 1996) Other studies
implicate the accumulation of extrachromosomal DNA as the cause of aging in yeast
(Sinclair and Guarente, 1997), and mutations in the yeast homolog of the Werner’s
Syndrome gene show premature aging. Oxygen free radicals have been proposed to
contribute to aging for many years (Harman, 1972), and more recent studies have shown
that overexpression of protective enzymes that break down oxygen radicals will extend the
life span of fruit flies (Orr and Sohal, 1994). Finally, decreased expression of cyclin-
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dependent kinases and increased expression of cyclin-dependent kinase inhibitors have
also been implicated as causes of proliferative arrest in cultured cells (Afshari and Barret,
1996).
Decreased lysosomal pathways of proteolysis may contribute to or be the result of these
causes of aging. For example, oxygen free radical damage may cause the reduced
macroautophagy, but reduced macroautophagy may also cause oxygen radical damage. If
inhibitors of the protective enzymes are normally degraded by macroautophagy, their
concentrations will increase with aging and the activities of the protective enzymes will
decrease. Additional studies are required to clarify these relationships.
7. Conclusions and future perspectives
Because of the nature of this review, we have mainly focused in the role of lysosomes
as proteolytic compartments but, as mentioned in the Introduction, lysosomes contain a
large variety of enzymes and therefore are involved in other many catabolic processes.
Age-related changes in these other lysosomal functions contribute to the final phenotype
of the “old” lysosomes. For example, a decrease in the activity of several lysosomal
glycosidases with age have been related with the accumulation of lipofucsin in the retina
epithelium (Cingle et al., 1996). Also, an abnormally increased ␤-galactosidase activity is
found in most senescent cells in culture and in aged skin and it is currently used as a
biomarker for senescence (Dimri et al., 1995). Finally, some regulatory molecules such as
vitamins and lipids are processed along the lysosomal system to become fully active.
Changes in the lysosomal system with age might affect the cellular processes mediated by
those compounds.
The diversity of intra- and extracellular proteolytic pathways that converge in the
lysosomal system are affected in very different ways in senescent organisms. Even for the
same lysosomal pathway, age-related changes depend on the tissue and cellular conditions
analyzed. The future analysis of each of the lysosomal functions separately and under
different cellular conditions in senescent organisms will certainly contribute to the understanding of the age-related changes in the lysosomal system
The identification and correction of specific age-related defects in the lysosomal system
will help to discriminate between causes or consequences of the proteolytic failure with
age. For example, we are currently trying to correct in senescent cells in culture the
abnormally low lysosomal levels of the receptor protein for the chaperone-mediated
autophagy. Once the normal lysosomal levels of lamp2a are restored, we first will analyze
if correcting the defect results in a recovery of the normal function of the pathway, and
second, how that affects levels of postranslational modifications in proteins and total
protein content of the cells, for example. Future identification of specific defects for each
of the different lysosomal proteolytic pathways will allow similar approaches.
References
Afshari, C. & Barret, J. (1996). Molecular genetics of in vitro cellular senescence. In N Holbrrok, G Martin &
R Lockshin (Eds). Cellular Aging and Cell Death (pp. 109 –121). New York: Wiley-Liss Inc.
Agarraberes, F., Terlecky, S.R., & Dice, J.F. (1997). An intralysosomal hsp70 is required for a selective pathway
of lysosomal protein degradation. J Cell Biol 137, 825– 834.
A. M. Cuervo, J. F. Dice / Experimental Gerontology 35 (2000) 119 –131
129
Araki, N., Ueno, N., Chacrabarti, B., Morino, Y., & Horiuchi, S. (1992). Immunochemical evidence for the
presence of advanced glycation end products in human lens proteins and its positive correlation with aging.
J Biol Chem 267, 10211–10214.
Bednarski, E., Ribak, C.E., & Lynch, G. (1997). Suppression of cathepsins B and L causes a proliferation of
lysosomes and the formation of meganeurites in hippocampus. J Neurosci 17, 4006 – 4021.
Bi, X., Pinkstaff, J., Nguyen, K., Gall, C., & Lynch, G. (1998). Experimentally induced lysosomal dysfunction
disrupts processing of hypothalamic releasing factors. J Comp Neurol 401, 382–394.
Borg, W., During, M., Sherwin, R., Borg, M., Brines, M., & Shulman, G. (1994). Ventromedial hypothalamic
lesions in rats suppress counterregulatory responses to hypoglycemia. J Clinical Invest 93, 1677–1682.
Bowers, W. (1998). Christian de Duve and the discovery of lysosomes and peroxisomes. Trends Cell Biol 8,
330 –333.
Carafoli, E. & Molinari, M. (1998). Calpain—a protease in search of function [Review]. Biochem Biophys Res
Comm 247, 193–203.
Choppra, V., Moozar, K., Mehindate, K., & Schipper, H. (1997). A cellular stress model for the differential
expression of glial lysosomal cathepsins in the aging nervous system. Exp Neurol 147, 221–228.
Chu, T., Tran, T., Yang, F., Beech, W., Cole, G., & Frautschy, S. (1998). Effect of chloroquine and leupeptin
on intracellular accumulation of amyloid-beta (AB) 1-42 peptide in a murine N9 microglial cell line. FEBS
Lett 436, 439 – 444.
Cingle, K., Kalski, R., Bruner, W., O’Brien, C., Erhard, P., & Wyszynski, R. (1996). Age-related changes of
glycosidases in human retinal pigment epithelium. Curr Eye Res 15, 433– 438.
Cuervo, A. & Dice, J. (1998). How do intracellular proteolytic systems change with age? Frontiers Biosci 3,
25– 43.
Cuervo, A. & Dice, J. F. (1996). A receptor for the selective uptake and degradation of proteins by lysosomes.
Science 273, 501–503.
Cuervo, A. & Dice, J. (1998). Lysosomes, a meeting point of proteins, chaperones, and proteases. J Mol Med
76, 6 –12.
Cuervo, A., Dice, J. F., & Knecht, E. (1997). A lysosomal population responsible for the hsc73-mediated
degradation of cytosolic proteins in lysosomes. J Biol Chem 272, 5606 –5615.
Dean, R., Stocker, S. F. R., & Davies, M. J. (1997). Biochemistry and pathology of radical-mediated protein
oxidation. Biochem J 324, 1–18.
DeMartino, G. & Slaughter, C. (1999). The proteasome, a novel protease regulated by multiple mechanisms
[Review]. J Biol Chem 274, 22123–22126.
Dice, J.F. (1982). Altered degradation of proteins microinjected into senescent human fibroblasts. J Biol Chem
257, 14624 –14627.
Dice J. F. (1993). Cellular and molecular mechanisms of aging. Physiol Rev 73, 149 –159.
Dice, J. F. (2000). Lysosomal Pathways of Protein Degradation Landes Bioscience, Austin, TX.
Dimri, G., Lee, X., Basile, G., et al. (1995). A biomarker that identifies senescent human cells in culture and in
aging skin in vivo. Proc Natl Acad Sci USA 92, 9363–9367.
Dini, L., Rossi, L., Marchese, E., Tuzittu, M., & Rotilio, G. (1996). Age-related changes in the binding and
uptake of Cu, Zn superoxide dismutase in rat liver cells. Mech Ageing Dev 90, 21–33.
Economou, A. & Wickner, W. (1994). SecA promotes preprotein translocation by undergoing ATP-driven cycles
of membrane insertion and deinsertion. Cell 78, 835– 843.
Frautschy, S., Horn, D., Sigel, J., Harris–White, M., Mendoza, J., Yang, F., Saido, T., & Cole, G. (1998).
Protease inhibitor coinfusion with amyloid ␤-protein results in enhanced deposition and toxicity in rat brain.
J Neurosci 18, 8311– 8321.
Gafni, A. (1997). Structural modifications of proteins during aging. J Am Geriatric Soc 45, 871– 880.
Glaser, T., Schwarz–Ben Meir, N., Barnoy, S., Barak, S., Eshhar, Z., & Kosower, N. (1994). Calpain (Ca⫹2dependent thiol protease) in erythrocytes of young and old individuals. Proc Natl Acad Sci USA 91,
7879 –7883.
Gracy, R., Talent, J., & Zvaigzne, A. (1998). Molecular wear and tear leads to terminal marking and the unstable
isoforms of aging. J Exp Zool 282, 18 –27.
Gurley, R. & Dice, J. F. (1988). Degradation of endocytosed proteins is unaltered in senescent human fibroblasts.
Cell Biol Int Rep 12, 885– 894.
Haas, C. & Mandelkow, E. (1999). Proteolysis by presenilins and the renaissance of tau. Trends Cell Biol 9,
241–246.
Harman, D. (1972). The biologic clock: the mitochondria? J Am Geriatr Soc 20, 145–147.
130
A. M. Cuervo, J. F. Dice / Experimental Gerontology 35 (2000) 119 –131
He, Y., Kwan, W., & Steinbrecher, U. (1996). Effects of oxidized low density lipoprotein on endothelin secretion
by cultured endothelial cells and macrophages. Atherosclerosis 119, 107–118.
Hochschild, R. (1971). Lysosomes, membranes and aging. Exp Gerontol. 6, 153–166.
Jadot, M., Wattiaux, R., Mainferme, F., Dubois, F., Claessens, A., & Wattiaux–De Coninck, S. (1996). Soluble
form of Lamp II in purified rat liver lysosomes. Biochem Biophys Res Comm 223, 353–359.
Jurivich, D., Qiu, L., & Welk, J.F. (1997). Attenuated stress responses in young and old human lymphocytes.
Mech Ageing Develop 94, 233–249.
Kominami, E., Ezaki, J.M., D, Ishido, K., Uenot, T., & Wolfe, L. (1992). Specific storage of subunit c of
mitochondrial ATP synthase in lysosomes of neuronal ceroid lipofuscinosis (Batten’s Disease). J Biochem
111, 278 –282.
Lee, H., Paz, M.A., & Gallop, P.M. (1982). Low density lipoprotein receptor binding in aging human diploid
fibroblasts in culture. J Biol Chem 257, 8912– 8918.
Lee, H. & Wei, Y. (1997). Mutation and oxidative damage of mitochondrial DNA and defective turnover of
mitochondria in human aging. J Formosan Med Assoc 96, 770 –778.
Li, K. & Greenwald, I. (1997). HOP-1 a Caenorhabditis elegans presenilin, appears to be functionally redundant
with SEL-12 p, appears to be functionally redundant with SEL-12 presenilin and to facilitate LIN-12 and
GLP-1 signaling. Proc Nat Acad Sci 94, 12204 –12209.
Makrides, S. (1983). Protein synthesis and degradation during aging and senescence. Biol Rev 83, 393– 422.
Malorni, W., Testa, U., Rainaldi, G., Tritarelli, E., & Peschle, C. (1998). Oxidative stress leads to a rapid
alteration of transferrin receptor intravesicular trafficking. Exp Cell Res 241, 102–116.
Martin, G., Austad, S., & Johnson, T. (1996). Genetic analysis of aging: role of oxidative damage and
evironmental stresses. Nature Genetics 13, 25–30.
Miller, R. (1999). Aging and Immune Function. In W. Paul (Ed), Fundamental Immunology (pp. 947–966).
Philadelphia: Lippincott-Raven Publishers.
Mizushima, N., Noda, T., Yoshimori, T., Tanaka, Y., Ishii, T., George, M.D., Klionsky, D.J., Ohsumi, M., &
Ohsumi, Y. A. (1998). Protein conjugation system essential for autophagy. Nature 395, 395–398.
Mortimore, G., Miotto, G., Venerando, R., & Kadowaki, M. (1996). Autophagy Biochem 27, 93–135.
Orr, W. & Sohal, R. (1994). Extension of life span by overexpression of superoxide dismutase and catalase in
Drosophila melanogaster. Science 263, 1128 –1130.
Paresce, D., Chung, H., & Maxfield, F. (1996). Slow degradation of aggregates of the Alzheimer’s disease
amyloid beta-protein by microglial cells. J Biol Chem 272, 29390 –29397.
Peterson, S., Klabunde, T., Lahuel, H., Purkey, H., Sacchettini, J., & Kelly, J. (1998). Inhibition transthyretin
conformational changes that lead to amyloid fibril formation. Proc Nat Acad Sci 95, 12956 –12960.
Prasanna, H. & Lane, R. (1979). Protein degradation in aged nematodes (Turbatrix aceti). Biochem Biophys Res
Comm 86, 552–559.
Reznick, A. & Gershon, D. (1979). The effect of age on the protein degradation system in the nematode
Turbatrix aceti. Mech Ageing Develop 11, 403– 415.
Saito, K., Elce, J.S., Hamos, J.E., & Nixon, R.A. (1993). Widespread activation of calcium-activated neutral
proteinase (calpain) in the brain in Alzheimer’s disease: a potential molecular basis for neuronal degeneration. Proc Natl Acad Sci USA 90, 2628 –2632.
Sakai, Y., Koller, A., Rangell, L., Keller, G., & Subramani, S. (1998). Peroxisome degradation by microautophagy in Pichia pastoris. Identification of specific steps and morphological intermediates. J Cell Biol 141,
625– 636.
Seglen, P., Gordon, P., & Holen, I. (1990). Nonselective autophagy. Semin Cell Biol 1, 441– 448.
Shang, F., Gong, X., Palmer, H.J., Nowell, T.R., & Taylor, A. (1997). Age-related decline in ubiquitin
conjugation in response to oxidative stress in the lens. Exp Eye Res 64, 21–30.
Shibatani, T. & Ward, W. (1996). Effect of age and food restriction on alkaline protease activity in rat liver. J
Gerontol 51, B316 –B322.
Sinclair, D. & Guarente, L. (1997). Extrachromosomal rDNA circles- A cause of aging in yeast. Cell 91,
1033–1042.
Sleat, D.E., Donnelly, R.J., Lackland, H., Liu, C.G., Sohar, I., Pullarkat, R.K., & Lobel, P. (1997). Association
of mutations in a lysosomal protein with classical late-infantile neuronal ceroid lipofuscinosis. Science 277,
1802–1805.
Subramani, S. (1998). Components involved in peroxisome import, biogenesis, proliferation, turnover, and
movement. Physiol Rev 78, 1–18.
A. M. Cuervo, J. F. Dice / Experimental Gerontology 35 (2000) 119 –131
131
Sukharev, S., Pleshakova, O., Moshinikova, A., Sadovnikov, V., & Gaziev, A. (1997). Age- and radiationdependent changes in carbonyl content, susceptibility to proteolysis, and antigenicity of soluble rat liver
proteins. Comp Biochem Physiol 116, 333–338.
Terman, A. (1995). The effect of age on formation and elimination of autophagic vacuoles in mouse hepatocyte.
Gerontology 41, 319 –325.
Terman, A. & Brunk, U. (1998). Lipofuscin—mechanisms of formation and increase with age [Review]. APMIS
106, 265–276.
Terman, A., Dalen, H., & Brunk, V.T. (1999). Ceroid/lipofuscin-loaded human fibroblasts show decreased
survival time and diminished autophagocytosis during amino acid starvation. Exp Gerontol 34, 943–957.
Tomkinson, B. (1999). Tripeptidyl peptidases: enzymes that count. Trends Biochem Sci 24, 355–359.
van den Hove, M., Couvreur, M., Authelet, M., & Neve, P. (1998). Age delays thyroglobulin progression
towards dense lysosomes in the cream hamster thyroid. Cell Tissue Res 294, 125–135.
Vetvicka, V., Tlaskalova–Hogenova, A., & Pospisil, M. (1985). Impaired antigen presenting function of
macrophages from aged mice. Immunol Invest 14, 105–114.
Vines, D. & Warburton, M. J. (1998). Purification and characterization of a tripeptidyl aminopeptidase I from
rat spleen. Biochim Biophys Acta 1384, 233–242.
Ward, W. & Shibatani, T. (1994). Dietary modulation of protein turnover. In B. Yu (Ed). Modulation of Aging
Processes by Dietary Restriction (pp. 121–142). Boca Raton, FL: CRC Press.
Werb, Z. (1997). ECM and cell surface proteolysis: regulating cellular ecology. Cell 91, 439 – 442.
Wright, W., Brassiskyte, D., Piatyszek, M., & Shay, J. (1996). Experimental elongation of telomeres extends the
lifespan of immortal x normal cell hybrids. EMBO J 15, 1734 –1741.
Yokota, S. (1993). Formation of autophagosomes during degradation of excess peroxisomes induced by
administration of dioctyl phthalate. Eur J Cell Biol 61, 67– 80.