Pathophysiology of chaperone-mediated autophagy

The International Journal of Biochemistry & Cell Biology 36 (2004) 2420–2434

Review

Pathophysiology of chaperone-mediated autophagy Ashish Massey, Roberta Kiffin, Ana Maria Cuervo∗ Department of Anatomy and Structural Biology, Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Ullmann Building Room 614, 1300 Morris Park Avenue, Bronx, NY 10461, USA

Abstract In contrast to the classically described “in bulk” lysosomal degradation, the first evidence for selective degradation of cytosolic proteins in lysosomes was presented more than 20 years ago. Throughout this time, we have gained a better understanding about this process, now known as chaperone-mediated autophagy (CMA). The identification of new substrates for CMA and novel components, in both the cytosol and the lysosomes, along with better insights on how CMA is regulated, have all helped to shape the possible physiological roles of CMA. We review here different intracellular functions of CMA that arise from its unique characteristics when compared to other forms of autophagy. In view of these functions, we discuss the relevance of the changes in CMA activity in aging and in different pathological conditions. © 2004 Elsevier Ltd. All rights reserved. Keywords: Lysosomes; Aging; Lysosomal storage disorders; Nephropathy; Neurodegeneration

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . What is different in CMA? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulation of CMA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Physiological role of CMA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CMA and aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CMA and disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. CMA and nephropathies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. CMA and lysosomal storage disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. CMA and oxidative injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4. CMA and neurodegeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. 2. 3. 4. 5. 6.

∗

Corresponding author. Tel.: +1-718-430-2689; fax: +1-718-430-8975. E-mail address: [email protected] (A.M. Cuervo).

1357-2725/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2004.04.010

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1. Introduction The degradation of cytosolic components by lysosomes is generically known as autophagy, a process conserved from yeast to mammalian cells (Cuervo, 2004; Dice, 2000; Wang & Klionsky, 2003). The two main steps in autophagy are the delivery of the substrates to the lysosomal lumen, followed by their breakdown inside lysosomes by resident enzymes. The fate of the substrates once inside the lysosomal lumen is invariable and irreversible, being degraded into their constituents with a half-life of 5–10 min (Dice, 2000; Meijer, 2003; Seglen & Bohley, 1992). In contrast with this uniformity in the degradation step, different mechanisms lead to the delivery of substrates into the lysosomal compartment, setting the basis for the classification of the different forms of autophagy. Although with some variations, two forms of autophagy, macroautophagy and microautophagy, are conserved throughout the phylogenetic scale (Huang & Klionsky, 2003). In mammalian cells, a third form of autophagy, chaperone-mediated autophagy (CMA), also contributes to the removal of cytosolic components by lysosomes (Cuervo, 2004; Dice, 2000; Wang & Klionsky, 2003). Autophagy plays an essential role in the maintenance of cellular homeostasis (Dice, 2000; Meijer, 2003; Seglen & Bohley, 1992). Along with the other major proteolytic systems inside the cells (i.e. proteasome/ubiquitin and calpains), autophagy is responsible for the continuous turnover of intracellular components, from single macromolecules (proteins, lipids, glycidic chains and nucleic acids) to whole organelles. Although autophagy is always active to some extent, maximal activation is attained under conditions leading to cellular stress (Dice, 2000; Meijer, 2003; Seglen & Bohley, 1992). Autophagy plays a defensive role by protecting cells against biological, physical and chemical injuries (Dorn, Dunn, & Progulske-Fox, 2002; Paglin et al., 2001; Suhy, Giddings, & Kirkegaard, 2000; Talloczy et al., 2002). This defense can be both direct, when harmful agents are targeted to lysosomes for destruction, or indirect, when autophagy takes care of eliminating the intracellular structures damaged during injury (Elmore, Qian, Grissom, & Lemasters, 2001; Lemasters et al., 2002; Tolkovsky, Xue, Fletcher, & Borutaite, 2002; Xue, Fletcher, & Tolkovsky, 2001). Autophagy is

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also essential for morphogenesis, tissue remodeling and cell differentiation (Hennig & Neufeld, 2002; Melendez et al., 2003; Otto, Wu, Kazgan, Anderson, & Kessin, 2003). This myriad of autophagic functions results from the diversity of the autophagic forms and the unique characteristics attributed to each of them. In this work, we review the physiological role of one of these autophagic pathways, CMA, and the possible relations between CMA malfunctioning and selected pathologies. Readers are referred to previous works and other reviews in this focused issue to learn about the pathophysiology behind the other autophagic forms (Cuervo, 2004; Mizushima, Ohsumi, & Yoshimori, 2002; Reggiori & Klionsky, 2002; Thumm, 2000).

2. What is different in CMA? Through macro- and microautophagy a region of the cytosol, including not only soluble proteins but also complete organelles, is sequestered and delivered for degradation by lysosomal enzymes (Fig. 1). In macroautophagy, sequestration requires “de novo” formation of a limiting membrane that surrounds the cytosolic region to be degraded and seals, forming an intermediate vesicle (autophagosome) (Mizushima et al., 2002; Ohsumi, 2001). After fusing with lysosomes, this vesicle acquires the lysosomal enzymes necessary to guarantee the complete degradation of the sequestered components. In the case of microautophagy, the lysosomal membrane itself sequesters the cytosolic region to be degraded. The substrates are thus internalized in the form of single membrane vesicles, or tubo-vesicular structures (Mortimore, Lardeux, & Adams, 1988; Sakai, Koller, Rangell, Keller, & Subramani, 1998). Once the limiting membrane of these vesicles is degraded, the lysosomal enzymes gain access to the internalized components. In these two forms of autophagy an entire region of cytosol is internalized inside lysosomes and consequently, all the cytosolic components in that area are degraded at the same time. In contrast, in CMA only selective cytosolic proteins are targeted and translocated, one at a time, into the lysosomal lumen (Cuervo & Dice, 1998; Dice, 2000). This distinctive mechanism of lysosomal transport determines the unique properties of CMA when compared to the other forms

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Fig. 1. Schematic model of the main forms of autophagy in mammalian cells. Internalization of complete regions of cytosol first into autophagosomes that fuse then with lysosomes (macroautophagy) or directly by the lysosomal membrane (microautophagy) contrast with the selective uptake in a molecule-by-molecule basis of cytosolic proteins via chaperone-mediated autophagy.

of autophagy (summarized in Fig. 2): only soluble proteins but not organelles are degraded through CMA; all CMA substrates contain a lysosomal targeting motif specific for this pathway; substrates bind to a protein/receptor at the lysososomal membrane; unfolding of substrates is required prior to translocation; binding/translocation are the rate limiting steps (Cuervo & Dice, 1998; Dice, 2000). The targeting motif of the CMA substrates is a series of five consecutive amino acids biochemically

related to the pentapeptide KFERQ (Dice, 1990). The Q residue is always located at the beginning or at the end of the sequence. The rest of the sequence contains a hydrophobic, a basic and an acid residue, along with a second hydrophobic or basic amino acid in any given order (Dice, 1990; Majeski & Dice, 2004). By sequence analysis, about 30% of the proteins in the cytosol contain this targeting motif and can be, in theory, substrates for CMA (Wing, Chiang, Goldberg, & Dice, 1991). A detailed list of already identified CMA

Fig. 2. Main characteristics of macroautophagy, microautophagy and chaperone-mediated autophagy. These characteristics refer to general macro- and microautophagy. Properties of some specialized variations of these two forms of autophagy, such as the one that selectively removes peroxisomes (pexophagy) can be found elsewhere (Bellu & Kiel, 2003; Kim & Klionsky, 2000).

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Fig. 3. Hypothetical model for chaperone-mediated autophagy. Substrates for CMA contain a targeting motif that is recognized by hsc70, a cytosolic chaperone that targets them to the lysosomal membrane. Substrates bind to lamp2a at the lysosomal membrane, are unfolded and get translocated into the lysosomal lumen assisted by the lysosomal chaperone lys-hsc70. Once in the lysosomal matrix substrates are rapidly degraded by the lysosomal proteases.

substrates and their targeting motifs can be found in the review by Dice and coworkers in this same Special Issue (Majeski & Dice, 2004). It is important to note that targeting motifs for different proteolytic systems often coexist in the same protein. Consequently, a single protein can be degraded through different pathways in the same cell depending on, among other factors, the state of the protein and the cellular conditions. The targeting motif for CMA is recognized in the cytosol by a molecular chaperone, which is the constitutive member of the hsp70 family of chaperones (hsc70) (Chiang, Terlecky, Plant, & Dice, 1989) (Fig. 3). The interaction between the substrate and hsc70 is modulated by other cochaperones and leads to the targeting of this complex to the lysosomal membrane. Treatment of the lysosomal membrane with different proteases, prior to exposure to substrates, revealed that protease-sensitive component(s) are required for substrate binding (Cuervo, Terlecky, Dice, & Knecht, 1994; Terlecky & Dice, 1993). To date, only a single transmembrane protein, the lysosome-associated membrane protein type 2a (lamp2a), has been shown to participate in substrate binding/uptake (Cuervo & Dice, 1996). CMA substrates directly interact with the cytosolic tail of lamp2a, resulting in the internalization of the substrates only, and not the cytosolic

chaperones. Substrates can bind lamp2a in their native conformation, however unfolding is required prior to transport (Salvador, Aguado, Horst, & Knecht, 2000). The chaperone/cochaperones associated with the substrate are likely to assist in the unfolding process (Agarraberes & Dice, 2001). Complete translocation of the substrate requires the presence of a resident lysosomal chaperone (lys-hsc70) (Agarraberes, Terlecky, & Dice, 1997; Cuervo, Dice, & Knecht, 1997). Similar to other chaperones that participate in protein import into other organelles, it is not yet clear whether lys-hsc70 acts by anchoring the substrate protein to prevent its retrograde movement to the cytosol, or if it actively pulls the protein into the lysosomal lumen. Like in the other forms of autophagy, once the substrate reaches inside the lysosome it is rapidly degraded.

3. Regulation of CMA CMA is a generalized form of autophagy described in many different cellular types and tissues (Cuervo & Dice, 1998; Dice, 2000). The CMA-related components identified to date—cyt-hsc70 and associated cochaperones, lamp2a and lys-hsc70—are also

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ubiquitously distributed in mammals (Gough & Fambrough, 1997; Lichter-Konecki et al., 1999; Sarafian et al., 1998). Despite this broad distribution, levels of these components vary depending on cell type and result in differences in CMA activity. Some level of CMA can be detected in organs such as liver, kidney, heart and spleen, even under basal conditions. However, maximal activation of CMA is attained in these organs under conditions of stress (i.e. starvation, pro-oxidant conditions, exposure to certain toxic compounds) (Cuervo, Knecht, Terlecky, & Dice, 1995b; Cuervo, Hildebrand, Bomhard, & Dice, 1999). In other tissues, such as skeletal muscle, CMA is not upregulated under any of those conditions (Wing et al., 1991). Nevertheless, since the basic components for CMA are also present in this tissue, we cannot disregard that under certain circumstances CMA could become active. The presence of lys-hsc70 in the lysosomal lumen is absolutely necessary for substrate uptake. In fact, activation of CMA during starvation is concomitant with an increase in the levels of lys-hsc70 inside lysosomes (Agarraberes et al., 1997; Cuervo et al., 1995b). Once levels of lys-hsc70 are enough to guarantee substrate translocation, regulation of CMA depends on the levels of lamp2a at the lysosomal membrane (Cuervo & Dice, 2000b). Whenever CMA activity increases, levels of lamp2a at the lysosomal membrane are higher, and vice versa. Even more, overexpression of lamp2a in cultured cells increases CMA activity at a rate proportional to the increase in lamp2a levels (Cuervo & Dice, 2000c). Changes in lamp2a levels are regulated by different mechanisms depending on cellular conditions and the stimuli that activate CMA. During nutritional starvation, the increase in the levels of lamp2a does not require de novo synthesis of the protein, but it is attained by preventing its normal degradation in the lysosomal compartment (Cuervo & Dice, 2000b). If starvation persists, part of the pool of lamp2a normally located in the lysosomal lumen is now recruited to the lysosomal membrane where it becomes active for substrate binding/uptake (Cuervo & Dice, 2000b). In contrast, in acute conditions leading to CMA activation, such as exposure to pro-oxidant agents, the increase in the levels of lamp2a results predominantly from de novo synthesis of the protein (R. Kiffin et al., submitted for publication).

The signals that induce activation of CMA and the transcriptional cascades that participate in this process have not yet been elucidated. The variety of conditions that activate CMA makes the existence of different activating signals likely. During nutrient deprivation, changes in the levels of ketone bodies circulating in blood are sufficient to activate CMA (J.F. Dice, P. Finn, personal communication). In kidney epithelial cells, the epidermal growth factor reduces CMA activity (Franch, Sooparb, Du, & Brown, 2001). Although secondary messengers for this inhibition have not been identified, functional Ras and class 1 PI 3-kinases are required (Franch, Wang, Sooparb, Brown, & Du, 2002). In contrast with these extracellular signals, activation of CMA during oxidative stress or selective protein damage is likely to result from an intracellular signal.

4. Physiological role of CMA In addition to the functions common to all forms of autophagy, the particular characteristics of CMA confer this pathway with unique functions too. The best studied role of CMA is during nutritional stress (Cuervo et al., 1995b; Terlecky, Chiang, Olson, & Dice, 1992). Activation of CMA in cultured cells and in rodent tissues correlates with their nutritional state, since removal of serum (in cells in culture) or prolonged starvation (in rodents) results in increased rates of CMA (Backer, Bourret, & Dice, 1983; Cuervo et al., 1995b; Neff, Bourret, Miao, & Dice, 1981; Wing et al., 1991). In contrast to microautophagy which is constitutively activated in almost all cells and tissues, macroautophagy, the other major form of autophagy, is also activated by nutrient deprivation (Meijer, 2003; Seglen & Bohley, 1992). How does activation of CMA and macroautophagy relate? Studies in rat liver have revealed that during the first hours of starvation only macroautophagy is activated (Meijer, 2003; Seglen & Bohley, 1992). A major intracellular kinase, mTOR acts as a nutrient sensor, modulating the downstream signaling cascades that regulate macroautophagy. In addition, in some types of cells, mTOR-independent activation is also possible (Mordier, Deval, Bechet, Tassa, & Ferrara, 2000). Morphological and metabolic measurements indicate that macroautophagy reaches maximal activation around 6 h after nutrient removal

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and then progressively decays. In fact, it is difficult to detect formation of new autophagic vesicles beyond 10–12 h of starvation (de Waal, Vreeling-Sindelarova, Schellens, Houtkooper, & James, 1986; Ueno, Muno, & Kominami, 1991). The time-course of activation of CMA overlaps only partially with macroautophagy, reaching maximal activity later on (Cuervo et al., 1995b). Increase in CMA activity is apparent after 8–10 h of starvation but, rates of CMA progressively increase up to 88 h of starvation. A similar pattern has been recently described for confluent fibroblasts in culture after serum removal (Fuertes, Martin De Llano, Villarroya, Rivett, & Knecht, 2003). In this case, after 4 h of serum deprivation macroautophagy is the predominant form of protein degradation, but after 12 h it only contributes to 30% of lysosomal protein breakdown, being progressively replaced by CMA. It is not clear how the sequential activation of macroautophagy/CMA is coordinated. One possibility is that the macroautophagic engulfment of cytosolic regions enriches lysosomes in some component(s) required for CMA. A good candidate would be hsc70, since two-dimensional electrophoretic studies have revealed that cytosolic and lysosomal hsc70 are recognized by the same antibodies and are probably the same protein, but only the most acidic isolectric form of the cytosolic hsc70 is located in the lysosomal lumen (Agarraberes et al., 1997). It is also possible that a still unknown physiological inhibitor of CMA is degraded by macroautophagy leading to progressive activation of CMA as cytosolic levels of the inhibitor decrease. Independent of the mechanism, this coordinated action between both pathways probably obeys the need of switching to a more selective degradation as starvation progresses. In the first stages of starvation, activation of macroautophagy provides the essential components (amino acids, lipids, glycidic groups and nucleotides) required to continue synthesis of essential macromolecules for the cell. However, if the starvation persists more than 6–8 h, nonselective degradation of cytosolic components may no longer be desirable, since it could lead to elimination of essential cellular components or components just synthesized to respond to the nutritional stress. Under these conditions, some selectivity of substrates would be preferable. In the case of proteins, CMA can provide that selectivity. Some of the proteins containing the CMA targeting

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motif could be proteins nonessential under those nutritional conditions (for example, several glycolytic enzymes, shown to be CMA substrates (Aniento, Roche, Cuervo, & Knecht, 1993), could be eliminated during starvation when glycolysis is reduced). Direct uptake of complex glycidic groups by lysosomes and release of simpler forms have been reported (Saint-Pol, Codogno, & Moore, 1999). Whether similar selective pathways also exist for degradation of complex lipids and nucleic acids is still unknown. It is also possible that the degradation of other KFERQ-containing proteins under these circumstances comply with a regulatory function. For example, the described degradation of some of the subunits of the 20S proteasome, the major cytosolic protease, by CMA could be responsible for the decrease in proteasome activity reported during nutrient starvation (Cuervo et al., 1995a). Since the proteasome is normally responsible for the degradation of important regulatory factors inside the cell, including many stress-related proteins, the decrease in the 20S proteolytic activity would prolong their half-life inside the cell. As new substrates for CMA are being described, new consequences of the activation of this pathway in the late stages of nutrient deprivation can be inferred. For example, CMA has been shown to be responsible for the degradation of a long-lived pool of the inhibitor of the nuclear factor ␬B (I␬B) inside some cells (Cuervo, Hu, Lim, & Dice, 1998). I␬B is the physiological inhibitor of the nuclear transcription factor NF␬B, and degradation of I␬B by the proteasome is a well-characterized mechanism for activation of this factor. After prolonged starvation, when proteasome activity is decreased, degradation of the long-lived I␬B by CMA could now be responsible for maintaining NF␬B-dependent transcription, required for stress management (Cuervo et al., 1998). Some of the possible consequences of CMA activation during prolonged stress are illustrated in Fig. 4. CMA is also activated in other stress conditions not directly related with the nutritional state, such as oxidation or exposure to selected toxic compounds (Cuervo et al., 1998, 1999). In both cases, damaged or altered proteins are present in the cytosol and are the likely signals that activate CMA. There is extensive literature supporting the removal of damaged or altered proteins by the ubiquitin/proteasome

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Fig. 4. Possible physiological roles for CMA. Activation of CMA during nutritional stress: (1) contributes amino acids for the synthesis of essential proteins by degrading nonessential ones; (2) preserves essential proteins substrate of the 20S proteasome by decreasing cytosolic levels of this protease; (3) and contributes to activation of transcription of stress-related proteins by degrading the physiological inhibitor of the NF␬B transcription factor. Activation of CMA during oxidative stress or other types of cell damage might be intended to selectively remove the damaged proteins sparing the intact ones. GAPDH: glyceraldehyde-3-phosphate dehydrogenase; PGM: phosphoglyceromutase; I␬B: inhibitor of the nuclear factor ␬B; ␣-2 mglob: alpha-2-microglobulin.

system (Grune, Merker, Sandig, & Davies, 2003; Imai, Yashiroda, Maruya, Yahara, & Tanaka, 2003), however, as mentioned before, the same protein can be degraded by different proteolytic systems depending on the cellular conditions. It is thus possible that CMA assists the proteasome/ubiquitin system in this function, and when required, takes over this task to avoid compromising proteasome function. The ability to selectively remove some proteins without altering others makes CMA more suitable as a replacement for the proteasome/ubiquitin system than other non-selective lysosomal pathways. Although there are reports of selectivity in degradation of organelles by macro- and microautophagy (Bellu & Kiel, 2003; Xue et al., 2001), evidence for selective degradation of soluble cytosolic proteins through these two pathways has not yet been found.

5. CMA and aging Total rates of protein degradation decrease with age (Bradley, Dice, Hayflick, & Schimke, 1975; Prasanna

& Lane, 1979; Reznick & Gershon, 1979). This age-related decrease is more pronounced when considering only the degradation of long-lived proteins that, with some exceptions, are degraded by lysosomes. A decrease with age in both macroautophagy and CMA has been reported (Cuervo & Dice, 2000a; Del Roso et al., 2003; Dice, 1982; Donati et al., 2001; Terman, 1995). The failure of macroautophagy in old organisms is in the formation of the autophagosomes and in their elimination, once formed, by fusion with lysosomes (Terman, 1995). The decrease of CMA with age has been shown for liver and kidney in old rodents, as well as for senescent cells in culture (Cuervo & Dice, 2000a; Dice, 1982). Separate analysis of the steps involved in degradation of substrates by CMA—chaperone binding, lysosomal targeting, membrane binding, uptake and degradation—revealed no changes in the chaperone’s ability to interact with the substrates and target them to the lysosomal membrane. The proteolytic activity of the lysosomal proteases was also found unchanged by age. The decrease in CMA activity with age is mostly due to reduced rates of binding

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and uptake of the substrates by lysosomes (Cuervo & Dice, 2000a). A decrease in the levels of lamp2a at the lysosomal membrane is behind the impaired CMA in aging. In fact, levels of lamp2a in rodent liver remain constant up to middle age and then gradually decrease with age. The decrease in the number of receptors per lysosome is initially compensated for by increasing the number of lysosomes committed to this pathway but, as age advances, this compensatory mechanism is not enough to maintain normal CMA activity in response to stress (Cuervo & Dice, 2000a). What causes the age-related decrease in lamp2a levels remains unknown. Growing evidence supports intercommunication among the different proteolytic systems, which are likely to be in a continuous balance. The same protein can be degraded by different proteolytic systems (Cuervo et al., 1998; Lenk, Susan, Hickson, Jasionowski, & Dunn, 1999); same components are shared by different forms of autophagy (Guan et al., 2001); blockage of one of the autophagic pathways is compensated, at least temporally, by activation of the others (Ding et al., 2003; Guan et al., 2001). However, since both macroautophagy and CMA are impaired with age, compensation is probably no longer possible and this failure could contribute to the decrease in the ability to remove damaged intracellular components and to adapt to stress, as seen in old organisms. Because of the selective character of CMA degradation, the malfunctioning of this pathway could also result in specific alterations in the pathways or cellular processes in which the substrates are involved. Only specific subunits of the 20S proteasome contain the KFERQ-related motif and are degraded by this pathway (Cuervo et al., 1995a). Different populations of 20S proteasome have been described based on their subunit composition (Farout, Lamare, Clavel, Briand, & Briand, 2003; Piccinini et al., 2003). Impairment of CMA with age could result in higher cytosolic levels of specific subunits of the 20S proteasome, and changes in the intracellular abundance of proteasome subpopulations. Whether these changes contribute to the age-dependent changes in the activity of the proteasome/ubiquitin system will require further investigation. The decrease of both macroautophagy and CMA with age can be prevented in animals subjected to caloric restriction, which is the only intervention

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known to slow down aging (Bergamini, Cavallini, Donati, & Gori, 2003; Cavallini, Donati, Gori, Pollera, & Bergamini, 2001; R. Kiffin, A.M. Cuervo, submitted for publication). In the case of macroautophagy, caloric restriction maintains the ability of the lysosomal system to respond to nutritional and hormonal changes even at advanced ages (Bergamini et al., 2003; Cavallini et al., 2001). Although the mechanism by which caloric restriction guarantees proper functioning of CMA in old rodents is not clear, it is interesting to point out that in these animals CMA is constitutively activated to values up to the ones detected after starvation in young rats (R. Kiffin, A.M. Cuervo, submitted for publication). This constitutive activation of CMA could contribute to the continuous removal of damaged proteins, thus preventing their accumulation inside cells. However, the long-term consequences of this continuous CMA activation will need further study.

6. CMA and disease Our current better understanding about the different physiological roles of CMA, as well as the identification of new substrates for this pathway, has contributed to linking CMA to different pathological conditions. In the following sections, we will review three conditions in which CMA activity is upregulated and one in which a decrease in CMA activity is likely to contribute to the etiopathogenesis of the disease. Up-regulation of CMA seems to be a defensive response in the two conditions in which protein damage occurs (a nephropathy induced by exposure to gasoline derivates and conditions leading to oxidative-stress). Up-regulation in the case of galactosialidosis, a lysosomal storage disease, is secondary to the genetic defect and explains some of the features accompanying this disorder. 6.1. CMA and nephropathies Although an increase in the levels of hsc70 inside lysosomes in kidney was noticed after acute tubular necrosis induced by inorganic mercury (Hernadez-Pando et al., 1995), the first direct connection between CMA and kidney pathologies was with the hyaline droplet nephropathy (Cuervo et al., 1999).

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Chronic exposure to environmental chemicals, including gasoline derivatives, results in the accumulation in the proximal tubules of hyaline droplets that lead to tubule necrosis in rats (Lehman-McKeeman et al., 1991). The selective damage in kidney, while other tissues are spared, is the consequence of the abundance in this organ of a lipoprotein, alpha-2-microglobulin (␣-2m), the main component of the hyaline droplets, that is a direct target for many of those compounds (Chaudhuri et al., 1999). ␣-2m is synthesized and secreted in liver, but it is mostly localized in kidney where it participates in the transport of fatty acids into renal epithelial cells. In both organs, a portion of the intracellular ␣-2m can be detected as a soluble protein in the cytosol. We noticed that ␣-2m contains a CMA-targeting motif in its sequence and that a percentage of intracellular ␣-2m (the cytosolic form) is normally degraded in lysosomes by CMA (Cuervo et al., 1999). Exposure to gasoline derivatives results in activation of CMA. The presence of the modified ␣-2m in the cytosol, or the toxic compound itself, may trigger the signal that normally activates CMA and facilitates the removal of the altered ␣-2m. As described for other conditions that lead to CMA activation, levels of lamp2a at the lysosomal membrane also increase in the toxic-exposed animals (Cuervo et al., 1999). It is likely that during the first stages of the nephropathy, activation of CMA is aimed to remove the damaged proteins from the cytosol in a selective manner. However, as exposure to the toxic compound continues this defensive mechanism is not enough, leading to the accumulation of the protein in kidney and the subsequent functional failure. Although the cytosolic proteolytic systems (i.e. calpains and the ubiquitin–proteasome pathway) play an integral role in kidney homeostasis under normal conditions, to date, only CMA has been implicated as a major contributor in regulating kidney growth (Franch, 2002). As in any other tissues, growth in kidney results from an unbalance in protein degradation/protein synthesis leaning toward protein synthesis. Franch et al. (2001) demonstrated that treatment of kidney cells with the growth factor EGF selectively decreases degradation of KFERQ-containing proteins. Among them, the increased levels of glycolytic enzymes previously identified as CMA substrates, point toward a raised need for energy as the cells undergo growth. In addition, this decline in CMA results in increased

intracellular levels of the paired box-related transcription factor Pax2, a KFERQ-containing protein that has been implicated in renal cell growth (Franch et al., 2001). Accompanying this diminished rate of degradation is a drop in the levels of lamp2a, suggesting a close link between protein accumulation (indicative of growing cells) and CMA. As mentioned before, the EGF-dependent changes in the receptor levels point to a novel mechanism of regulation of CMA. EGF suppresses proteolysis by a mechanism that involves Ras and class 1 PI 3-kinase (Franch et al., 2002). This role of CMA in the regulation of kidney growth may become relevant in pathological conditions such as diabetes mellitus, unilateral nephrectomy or chronic kidney damage which have been associated with an increase in cellular protein levels (hypertrophy). Although direct linkage to CMA has not yet been established, levels of the CMA substrate Pax2 increase during both kidney development and in renal cell carcinoma. Therefore, a correlation to CMA may be forthcoming (Franch et al., 2001). 6.2. CMA and lysosomal storage disorders Lysosomal storage disorders (LSD) is the generic name for genetic diseases resulting from alterations in the degradation of different types of macromolecules inside lysosomes (Neufeld, 1991; Wenger, Coppola, & Liu, 2002). In some of them the failure is in a specific enzyme, while in others the failure is in the mechanisms that deliver the substrates into lysosomes. A defect in the enzyme can result from alterations in its catalytic site or in its intracellular trafficking, which prevents it from reaching the lysosomal compartment (Mach, 2002). Although for each disease the substrate that accumulates inside lysosomes is different, the consequences of this accumulation are similar: enlargement of the lysosomal compartment, failure of other lysosomal functions, and often, leakage of lysosomal contents into cytosol and/or extracellular medium. Although most of the classically known LSDs originate from defects in glycases and lipases, recent studies have revealed a similar phenotype when specific lysosomal proteases (cathepsins) are knocked down. In fact, the cathepsin D knock-out has allowed the identification of mutations in this enzyme as the genetic defect in some forms of Batten Disease, a long known LDS (Dawson & Cho, 2000). It is likely

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that the modifications that the lysosomal compartment undergoes in these types of diseases interfere with normal lysosomal functioning, including CMA. The only LSD in which alterations in CMA have been demonstrated is galactosialidosis. In this case, not a decrease, but an increase in CMA activity has been reported. Enhanced CMA in galactosialidosis results from abnormally higher levels of lamp2a at the lysosomal membrane in the cells from these patients (Cuervo, Mann, Bonten, d’Azzo, & Dice, 2003). Cathepsin A/protective protein, the enzyme defective in this disorder, is required to stabilize two glycosidases in the lysosomal compartment (van der Spoel, Bonten, & d’Azzo, 1998). The absence or defect in cathepsin A leads to the defective functioning of the two enzymes and the intralysosomal accumulation of their substrates. In addition, cathepsin A participates in the normal degradation of lamp2a at the lysosomal membrane through its serine protease activity (Galjart et al., 1991). In fibroblasts from galactosialidosis patients or from cathepsin A knock-out mice, the half-life of lamp2a is abnormally prolonged resulting in constitutive activation of CMA (Cuervo et al., 2003). This continuous degradation could be behind the observed loss of weight and asthenia in these patients. Interestingly, restoration of normal levels of cathepsin A in cultured cells, by enzyme replacement, or in the animal model, through gene therapy, promotes degradation of lamp2a and returns CMA to normal levels (Cuervo et al., 2003). Recently, a defect in lamp2 has been shown to be the primary deficiency of Danon disease, a lysosomal glycogen storage disorder with normal acid maltase activity (Saftig, Tanaka, Lullmann-Rauch, & von Figura, 2001). Unfortunately, the interpretation of the phenotype and its relationship with altered CMA is not straightforward, since not only lamp2a, but also the other two lamp2 splicing variants (lamp2b and lamp2c) are defective in the affected patients. The consequences of impaired CMA in this disease would become clear once the intracellular role of each of the lamp2 isoforms is elucidated. 6.3. CMA and oxidative injury Oxidation is a form of stress extensively studied in the context of intracellular protein degradation. Much evidence supports oxidative stress’s role in initiation

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and progression of different pathologies (neurodegeneration, inflammatory lung diseases, diabetes, rheumatoid arthritis, artherosclerosis, motor neuron disorders, among others) and also in aging (Kohen & Nyska, 2002; Brune, Zhou, & von Knethen, 2003). Macroautophagy seems to play a major role in the elimination of organelles, predominantly mitochondria, damaged by free radicals (Elmore et al., 2001; Tolkovsky et al., 2002). There is vast literature supporting soluble cytosolic proteins being degraded by the proteasome system when exposed to oxidative damage (Grune et al., 2003). In vitro oxidation of some proteins increases their susceptibility to degradation by the 20S proteasome. It is believed that oxidation-induced conformational changes expose hydrophobic patches on the surface of the protein, which are recognized by the 20S proteasome, making ubiquitination unnecessary (Shringarpure, Grune, Mehlhase, & Davies, 2003). However, recently an ubiquitin ligase (E3) that selectively recognizes a protein when oxidized by iron has been described (Iwai, 2003). This suggests that selective ubiquitinization of oxidized proteins could also be used to target them to the proteasome system under some conditions. Oxidation of CMA substrates, both in vivo and in vitro, also increases their susceptibility to degradation by this pathway. Exposure of GAPDH to an iron-based oxidizing solution enhances its uptake by isolated lysosomes (A.M. Cuervo, E. Knecht, unpublished results). In Jurkat cells, treatment with antioxidants reduces the rate of degradation of the long-lived pool of I␬B by CMA (Cuervo et al., 1998). Degradation of the short-lived pool of I␬B by the proteasome also relates to its oxidative state (Piette et al., 1997). Neither phosphorylation nor ubiquitination, required for the degradation of I␬B by the proteasome, are necessary for lysosomal targeting (Cuervo et al., 1998). Added to the increased susceptibility of CMA substrates to be taken up by lysosomes when oxidized, we have found recently that oxidizing conditions activate CMA (R. Kiffin et al., submitted for publication). Lysosomes isolated from livers of mice exposed to paraquat, a pro-oxidant pesticide, take up more efficiently CMA substrates than lysosomes from untreated animals. Thus, both oxidation-induced conformational changes in soluble cytosolic proteins, as well as increased lysosomal activity, contribute to the increased rates of CMA during oxidative stress.

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How oxidation facilitates uptake of CMA substrates remains unknown. It is possible that oxidation-induced conformational changes expose hidden KFERQ-like sequences and facilitate hsc70 binding and lysosomal targeting. An intriguing idea proposed by Gracy, Talent, and Zvaigzne (1998) is that some covalent modifications, such as the ones that accumulate in aged cells, might transform a protein into a CMA substrate. These modifications or “terminal markings”, could be caused not only by oxidation, but also by deamidation or amino acid conversion. Conformational changes, which a protein undergoes as it is used (i.e. “molecular wear and tear”), increase the probability of such adjustments occurring. It is unclear as to whether or not these modified proteins can be repaired in vivo; though if they are, repair is limited. These changed proteins are more likely to be substrates of proteases, since they are unstable and unfold easier. Grazy et al. hypothesize that some of the modifications in the proteins could result in development of a KFERQ-like motif that will target them for degradation by CMA. For example, if a positive histidine gets oxidized it will behave as a negative aspartic acid residue, and it could complete a KFERQ-motif (Gracy et al., 1998). 6.4. CMA and neurodegeneration Alterations in protein degradation are common to several neurodegenerative diseases (Jellinger & Stadelmann, 2000; Larsen & Sulzer, 2002; Yuan, Lipinski, & Degterev, 2003). Decreased proteasome activity has been described in Parkinson’s and Huntington’s disease (Ciechanover & Brundin, 2003; Moore, Dawson, & Dawson, 2003). Autophagic vacuoles containing neuromelanin and lipofuscin can be found in aged brains and increase in degenerating neurons. Accumulation of these undigested products diminishes autophagocytic capacity (Brunk & Terman, 2002). Abnormal accumulation of autophagic vacuoles can be observed in the affected neurons in Alzheimer’s, Parkinson’s and Huntington’s disease (Cataldo, Hamilton, Barnett, Paskevich, & Nixon, 1996; Kegel et al., 2000; Nixon, Cataldo, & Mathews, 2000; Qin et al., 2003). It is unclear whether the increased number of autophagic vacuoles reflects a defensive response from the cells to eliminate the abnormal proteins that aggregate and accumulate in the neurons affected by those diseases, or if autophagic

vacuoles accumulate because of a primary defect in their fusion with lysosomes. Interestingly, three of the proteins found to be mutated in neurodegeneration—APP (for Alzheimer’s disease), synuclein (for Parkinson’s disease) and huntingtin (in Huntington’s disease)—contain KFERQlike motifs in their amino acid sequence, and could be, in theory, CMA substrates. So far, degradation by CMA has only been tested for synuclein (A.M. Cuervo et al., submitted for publication). A portion of the intracellular synuclein is normally degraded by CMA. However, mutations in synuclein described in some forms of Parkinson’s disease, decrease their uptake by lysosomes. Surprisingly, the mutated proteins show higher affinity for lamp2a binding, thus blocking uptake of other CMA substrates. The reasons for the increased affinity are currently unknown, but the decreased degradation of the mutated proteins and other CMA substrates is likely to increase their cytosolic levels and facilitate their aggregation.

7. Concluding remarks CMA allows selective degradation of soluble cytosolic proteins. This selectivity sets the tone for the physiological roles of this autophagic pathway. The ability of removing only tagged proteins allows CMA to discriminate between proteins located in the same cytosolic region, and resembles degradation by other nonlysosomal systems such as the ubiquitin/proteasome system. In addition, just as for other forms of autophagy, degradation of the substrates once internalized in the lysosomal lumen is rarely limiting, thus conferring this system with a large capacity comparable to other lysosome-mediated forms of degradation. In that sense, CMA could compensate, to some extent, for loss of activity in other intracellular proteolytic systems. Preferential activation of CMA under stress conditions places this pathway as part of the cellular protective response. Hyperactivation of CMA seems to have a beneficial effect in those conditions involving protein damage (i.e. toxic-induced cellular damage or oxidative stress). Malfunctioning of CMA can promote cytosolic accumulation/aggregation of specific proteins and could explain the impaired response to stress of old organisms.

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Although our knowledge about the possible physiological roles of CMA has improved and the consequences of changes in its activity are now better understood, there are still many questions that require further investigation. Would it be possible to restore normal CMA activity in old tissues by correcting the decrease in lamp2a levels? Could activation of CMA prevent the accumulation of neurodegeneration-related proteins containing a KFERQ motif? Other autophagic forms have been shown to play an important role in cancer progression (Ogier-Denis & Codogno, 2003; Paglin et al., 2001), but there is no information about CMA activity in tumor cells. Several pathogens selectively block macroautophagy in order to guarantee their survival inside the host cells (Dorn et al., 2002; Suhy et al., 2000; Talloczy et al., 2002). Is CMA hyperactivated in those cells? Failure of macroautophagy is a common feature in a group of muscular disorders, but nothing is known about how other autophagic pathways, including CMA, respond to that blockage. Some of these questions will find an answer as new CMA substrates are identified, better experimental tools are developed and our knowledge on the regulation of CMA advances.

Acknowledgements We would like to gratefully acknowledge Dr. Fernando Macian for critically reviewing this manuscript and the members of our laboratory for their valuable suggestions. Research in our laboratory is supported by National Institutes of Health/National Institute of Aging grants AG021904 and AG19834, a Huntington’s Disease Society of America Research grant and an Ellison Medical Foundation Award.

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