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 con- tribute 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) 0531-5565/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S0531-5565(00)00075-9 120 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 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 extra- cellular 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, neurode- generative 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