When Lysosomes Get Old૾

Experimental Gerontology 35 (2000) 119–131 Review When lysosomes get old૾

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

૾This work was supported by the National Institute of Aging AG008290 (A.M.C.) and AG06116 (J.F.D.). * Corresponding author. Tel.: ϩ617-636-6707; fax: ϩ617-636-0445. E-mail address: [email protected] (J. Dice)

Table 1. Effect of age on total protein degradation in different organs in rats

Organ Age Protein degradation Studies (young-old, months) (% decrease with age) (number) Whole body 1–20 56.1 Ϯ 3.1 4 Liver 6–22 50.0 Ϯ 4.1 4 Heart 1.5–12 28.3 Ϯ 0.9 4 12–24 46 1 0.8–26 62.5 Ϯ 2.2 2 Skeletal muscle Red 0.8–25 64.4 Ϯ 12.2 11 White 5–25 Ϫ53.7 1 Lung 0.8–25 0 3 Kidney 0.8–24 44.3 1 Brain 12–24 88.2 Ϯ 0.7 2

Data presented are modified from (Ward & Shibatani, 1994). Values are the medium Ϯ 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 summa- rized 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 122 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 ϭ Absorptive ϭ Receptor mediated ϭ, 2 Crinophagy 1 Macroautophagy 2 Chaperone-mediated autophagy 2 Microautophagy ?

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 inter- nalization, 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) 119–131 123

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 sup- pressed 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. 124 A. M. Cuervo, J. F. Dice / Experimental Gerontology 35 (2000) 119–131

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 126 A. M. Cuervo, J. F. Dice / Experimental Gerontology 35 (2000) 119–131

(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, phosphor- ylation, 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 undi- gested 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 A. M. Cuervo, J. F. Dice / Experimental Gerontology 35 (2000) 119–131 127 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 accumu- lates 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 metallopro- teases, 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- 128 A. M. Cuervo, J. F. Dice / Experimental Gerontology 35 (2000) 119–131 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 un- derstanding 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.

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