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J. Biol. Chem., Vol. 275, Issue 40, 31505-31513, October 6, 2000

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Age-related Decline in Chaperone-mediated Autophagy^*

Ana Maria Cuervo and J. Fred Dice

From the Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111

Received for publication, March 14, 2000, and in revised form, May 4, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Intracellular protein degradation rates decrease with age in many tissues and organs. In cultured cells, chaperone-mediated autophagy, which is responsible for the selective degradation of cytosolic proteins in lysosomes, decreases with age. In this work we use lysosomes isolated from rat liver to analyze age-related changes in the levels and activities of the main components of chaperone-mediated autophagy. Lysosomes from "old" (22-month-old) rats show lower rates of chaperone-mediated autophagy, and both substrate binding to the lysosomal membrane and transport into lysosomes decline with age. A progressive age-related decrease in the levels of the lysosome-associated membrane protein type 2a that acts as a receptor for chaperone-mediated autophagy was responsible for decreased substrate binding in lysosomes from old rats as well as from late passage human fibroblasts. The cytosolic levels and activity of the 73-kDa heat-shock cognate protein required for substrate targeting to lysosomes were unchanged with age. The levels of lysosome-associated hsc73 were increased only in the oldest rats. This increase may be an attempt to compensate for reduced activity of the pathway with age.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

One of the common characteristics of senescent cells is the accumulation of abnormal proteins in the cytosol (1). The described protein alterations are mostly post-translational modifications (2) and probably result from the inevitable exposure to damaging agents such as oxygen and glucose. All cells have specific mechanisms to eliminate damaged proteins (3-5). Age-related defects in intracellular proteolytic systems have been proposed to cause the accumulation of abnormal proteins (6). This hypothesis was initially supported by the fact that overall rates of protein degradation decline with age (1, 7).

Several different intracellular proteolytic systems contribute to total rates of protein degradation including the ubiquitin-proteasome system, a group of calcium-dependent proteases or calpains and lysosomes. Lysosomes are the proteolytic system most affected by age (8). The amount of ubiquitinated protein increases in "old" tissues, but its degradation rate does not significantly change with age (9). Some of the proteolytic activities of the 20 S proteasome, the core of the 26 S proteasome responsible for the degradation of ubiquitinated and some nonubiquitinated proteins, change with age, but the overall proteolytic rate is not altered (10). An increase rather than a decrease in calcium-dependent degradation of proteins with age has been reported in different tissues (11).

Proteins can be transported to lysosomes for degradation following several different pathways. The best characterized are endocytosis, crinophagy, macroautophagy, microautophagy, and chaperone-mediated autophagy (3, 12). Not all these lysosomal pathways of proteolysis are equally affected by age. A decrease of macroautophagy, chaperone-mediated autophagy, and some forms of endocytosis (receptor-mediated endocytosis) occurs in most tissues of old organisms (7, 13, 14).

Chaperone-mediated autophagy is activated in many types of confluent cells in culture during serum deprivation and in several tissues (liver, kidney, heart, spleen) from whole animals after prolonged starvation (15-17). Under those conditions, chaperone-mediated autophagy accounts for the degradation of 30% of cytosolic proteins. The identified substrates for this pathway include some glycolytic enzymes, transcription factors and their regulatory proteins, cytosolic proteases, cytosolic forms of secretory proteins, and lipid- and calcium-binding proteins (3, 12). All substrate proteins contain in their sequence a motif biochemically related to the pentapeptide KFERQ that targets the proteins for this pathway of lysosomal degradation (18). That motif is recognized by a molecular chaperone, the 73-kDa heat-shock cognate protein (hsc73),¹ which interacts with the substrate protein in the cytosol (19, 20). The chaperone-substrate protein complex binds to the lysosomal membrane through a receptor protein, the lysosome-associated membrane protein type 2a (lamp2a) (21). Once bound to the lysosomal membrane, the substrate protein is transported into the lysosomes assisted by another chaperone, the lysosomal hsc73 (ly-hsc73) that is normally present in the lysosomal matrix (22, 23). No transport of substrates takes place if ly-hsc73 is absent or experimentally blocked by endocytosis of specific antibodies. The levels of ly-hsc73 increase after nutrient deprivation and contribute to the increased chaperone-mediated autophagy rates under those conditions (17, 20). The level of the receptor at the lysosomal membrane is the rate-limiting step in protein binding and uptake (12, 21). This is regulated by changes in the lysosomal degradation of lamp2a and in its dynamic distribution between the lysosomal membrane and matrix, where a portion of intact lamp2a is located (24).

We have previously reported a decrease in chaperone-mediated autophagy using confluent cultures of senescent human fibroblasts (7, 25). In this study, we show that the decrease in chaperone-mediated autophagy with age is also evident using lysosomes isolated from rat liver. In addition, we have identified the effects of age on the various steps required for the degradation of proteins by this autophagic pathway. In both rat liver and human fibroblasts, the amount of lamp2a on the lysosomal membrane decreases with age.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Animals and Cells-- Male Fisher-344 rats of ages 3, 9, and 22 months were obtained from the age-controlled pool of animals maintained at the National Institutes of Health. Except where indicated, animals were starved for 20 h with free access to water before sacrifice to deplete liver glycogen that interferes with subcellular fractionation. Human lung fibroblasts (IMR-90) from the Coriell Cell Repositories (Camden, NJ) were maintained in Dulbecco's modified Eagle's medium (Sigma) in the presence of 10% newborn calf serum. To deprive cells of serum, plates were extensively washed with Hanks' balanced salts solution (Life Technologies, Inc.) and medium without serum was added.

Chemicals-- Sources of chemicals and antibodies were as described previously (17, 20, 21, 26). The antibody against the cytosolic tail of rat lamp2a was raised in our laboratory (21). The monoclonal antibody against the matrix side of rat lamp2s was kindly supplied by Dr. Michael Jadot (Facultes Universitaires Notre-Dame de la Paix, Namur, Belgium). The polyclonal antibodies against rat cathepsin L and lamp1 were generous gifts from Dr. Gary Sahagian (Tufts University, Boston, MA) and Dr. Ira Mellman (Yale University, New Haven, CT), respectively. The polyclonal antibody against cathepsin D was from Santa Cruz Biotechnologies (Santa Cruz, CA). The monoclonal antibodies against human lamp1 and lamp2 were obtained from the Developmental Studies Hybridoma Bank (Iowa City, IA). Gold-conjugated antibodies were from Goldmark Biologicals, Phillipsburg, NJ.

Isolation of Subcellular Fractions-- Rat liver lysosomes were isolated from a light mitochondrial-lysosomal fraction in a discontinuous metrizamide density gradient (27) by the shorter method reported previously (28). After isolation, lysosomes were resuspended in 0.3 M sucrose, 10 mM MOPS buffer. Integrity of the lysosomal membrane after isolation was systematically measured by -hexosaminidase latency as described previously (20). Only preparations with more than 95% intact lysosomes were used. Lysosomal matrices and membranes were obtained after hypotonic shock as described by Oshumi (29). Cytosolic fractions were obtained by centrifugation of the supernatant of the light mitochondrial-lysosomal fraction at 150,000 × g for 1 h at 4 °C. Lysosomes and lysosomal membranes from cultured cells were prepared as described previously (20).

Electron Microscopy, Morphometry, and Immunogold Procedures-- For conventional electron microscopy isolated lysosomes were double fixed (glutaraldehyde and OsO₄), embedded in Vestopal W, and stained with lead citrate as described previously (28). Ultrathin sections were observed in a Philips CM-10 electron microscope. Morphometric analysis was performed in randomly selected electron micrographs at a final magnification of × 25,000-35,000. Diameters and areas of the lysosomal profiles were measured in the electron micrographs. Lysosomal perimeters were determined by extrapolation of lysosomal profiles to oval shapes using the maximal and minimal diameters of each lysosome. Immunogold procedures were performed as described previously (17) using the antibodies against hsc73 or lamp2a followed by anti-mouse IgM or anti-rabbit IgG 10-nm gold conjugates. After incubation with the antibodies, the sections were stained with 5% uranylacetate for 15 min and with lead citrate for 20-25 s. Appropriate controls using only the gold-conjugated secondary antibodies were included. Localization of the gold particles on the lysosomal membrane or matrix was determined as described before (17), and the percentage of labeled lysosomes for a specific antibody was calculated by counting the labeled and unlabeled lysosomes among 500 lysosomes for each compared group.

Uptake and Degradation of Substrate Proteins by Isolated Lysosomes-- Substrate proteins were incubated with chymostatin-treated lysosomes as described previously (26, 28). Transport was measured after proteinase K treatment of the samples, SDS-PAGE, and immunoblot analysis, as the amount of substrate resistant to the protease. Degradation of substrate proteins by isolated intact lysosomes or lysosomal matrices was measured as described previously (20). We found an increase in the percentage of lysosomal breakage, measured as -hexosaminidase activity in the extralysosomal medium, in lysosomes from old rats only after a 90-min incubation at 25 °C. By decreasing the incubation time to 1 h (instead of the standard 2 h), we found that the levels of intact lysosomes at the end of the incubation were >95% in both age groups. GAPDH and RNase S-peptide were radioactively labeled by reductive methylation using [¹⁴C]formaldehyde or [³H]sodium borohydride (30). Substrates were incubated for 1 h at 25 °C with intact lysosomes. Reactions were stopped by the addition of trichloroacetic acid to a final concentration of 10%. Acid-soluble material (amino acids and small peptides) was collected by filtration through a Millipore multiscreen assay system (Millipore, Bedford, MA) using a 0.45-µm pore filter, and the acid-precipitable material (protein) was collected on the filter. Radioactivity in the samples was converted to dpm in a P2100TR Packard liquid scintillation analyzer by correcting for quenching using an external standard (Packard Instruments). Proteolysis was expressed as a percentage of the initial acid-insoluble radioactivity converted to acid-soluble radioactivity at the end of the incubation.

Substrate Binding Assays-- Binding of substrates to lamp2a was analyzed as described previously (21). Briefly, lysosomal membranes or matrices (100 µg of protein) were subjected to SDS-PAGE and then electrotransferred to a nitrocellulose membrane. The membrane was blocked with 5% nonfat milk in TTBS (20 mM Tris-HCl, pH 7.6, 137 mM NaCl, 0.1% Tween 20) and then incubated with the radiolabeled substrate in renaturation buffer (50 mM Tris-HCl, pH 7.5, 0.1 M potassium acetate, 0.15 M NaCl, 1 mM dithiothreitol, 5 mM MgCl₂, 1 mM EDTA, and 0.3% Tween 20) at 4 °C for 12 h. After extensive washing, the bound protein was detected by exposure to a phosphorimager screen. Binding of substrate proteins to hsc73 was analyzed using binding assay 2 described previously (31). hsc73 (5 µg) purified from the cytosol of young and old rat livers was precoated on 96-well microtiter plates and then incubated with [¹⁴C]GAPDH in TBS. After extensive washing the bound radiolabeled protein was eluted with 0.1% SDS and quantified by liquid-scintillation procedures as described above. Nonspecific binding was determined in a well precoated with ovalbumin, and that value was subtracted from all the experimental samples.

General Methods-- Cellular fractions and isolated proteins were subjected to SDS-PAGE in slab gels (32). Immunoblotting was performed following standard procedures using chemiluminescence methods (Renaissance^R, NEN-Life Science Products, Boston, MA) to visualize the peroxidase staining of the secondary antibodies. Protein concentration was determined by the Lowry method (33) using bovine serum albumin as a standard. Standard procedures were used for the determination of enzymatic activities as reported (20, 26, 28). hsc73 was purified from rat liver cytosol by ATP-agarose affinity chromatography (34), and where indicated the protein was radioactively labeled by reductive methylation as described above (30). Densitometric quantification of the immunoblotted membranes was performed on Kodak scientific imaging film, using an Image Analyzer System (Inotech S-100, Sunnyvale, CA). Statistical analyses were carried out using the Student's t test. Best fit straight lines were calculated by linear regression, and best fit curved lines and K_m and V_max values were calculated using Enzfitter software (Elsevier-Biosoft, Cambridge, UK).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Biochemical and Morphological Characteristics of Liver Lysosomes Isolated from Young and Old Rats-- The protein content and activities of different lysosomal, mitochondrial, and cytosolic marker enzymes in the lysosomal fractions isolated from livers of young (3-month-old) and old (22-month-old) rats are summarized in Table I. Lysosomes from old rats had higher protein content mainly because of increased matrix proteins. The total activity of two hydrolytic lysosomal enzymes (-hexosaminidase and -N-acetylglucosaminidase) was higher in lysosomes from old rats, but the specific activity of both enzymes was lower. A higher content of nonlysosomal proteins in old rat lysosomes could explain those results. Despite their higher content of nonlysosomal proteins, the activity of the two cathepsins analyzed, cathepsin A and B, was slightly higher in old rats.

The increase in nonlysosomal proteins in lysosomes from old rats was not caused by the lower purity of those preparations because the activity of mitochondrial and cytosolic markers were similar in both groups (Table I). The activity of some markers may change with age, so we also analyzed the lysosomal fractions for other contaminating organelles using electron microscopy. We observed less than 1 mitochondrion per 250 lysosomes in the preparations from young animals and about 2.3 mitochondria per 250 lysosomes in the preparations from old animals. However, the number of total contaminating mitochondria was similar in both groups when we discount the ones contained inside autophagic vacuoles (Fig. 1A, right, arrow).

View larger version (96K): [in this window] [in a new window]   Fig. 1.   Ultrastructure of liver lysosomes from young and old rats. A, lysosomes isolated from livers of 3-month-old (left) and 22-month-old (right) rats were processed for electron microscopy as described under "Experimental Procedures." A representative electron micrograph for each group is shown. OV, oval lysosomes with uniform content; RS, ring shaped lysosomes; IH, irregular lysosomes with heterogeneous content; bar, 0.5 µm; arrow, mitochondrion within an autophagic vacuole. B, the maximum and minimum lysosmal diameter was measured in 6-7 electron micrographs similar to those shown here (200 lysosomes for each age group). For each micrograph the perimeter (top) and areas (bottom) of every lysosome calculated by extrapolation to the closest ellipsoid shape are plotted. The mean value is indicated by the line.

Dramatic morphological changes of the lysosomal system in old rodents have been previously described in liver sections (35, 36). We have previously characterized the lysosomes active for chaperone-mediated autophagy and optimized the conditions to purify a lysosomal fraction enriched in those lysosomes (17, 23, 26, 28). We used electron microscopy procedures (Fig. 1A) to compare lysosomes isolated from livers of young and old rats. The different types of lysosomes and their contribution to the total lysosomal content in both age groups are summarized in Table II. In both groups, the lysosomal preparation consisted mainly of round and oval forms. The next most frequent form were lysosomes containing a single large electron-translucent vesicle or "ring shaped lysosomes" (as labeled in Fig. 1A). The number of lysosomes containing recognizable structures was slightly higher in the lysosomes isolated from old animals, most often corresponding to mitochondria (see Fig. 1A, right, arrow). Lipofuscin-like electron dense deposits were not significantly more abundant in the lysosomes from old animals when compared with young animals. We also calculated the perimeter and area of lysosomes, which were similar in both groups of animals (Fig. 1B). In summary, we did not find significant changes in the morphology, average size, or heterogeneity between the lysosomes isolated from livers of young and old rats.

We analyzed the protein content in the lysosomes isolated from young and old rat livers by SDS-PAGE (Fig. 2A) and immunoblot with specific antibodies against lysosomal membrane (Fig. 2B) and matrix (Fig. 2C) proteins. The protein pattern was similar in both age groups, but the abundance of some proteins changed with age. Lysosomal membrane proteins of 147, 95, 68, 62, 56, 32, and 30 kDa increased with age (Fig. 2A, solid arrowheads) as did matrix proteins of 147, 60, 45, 40, and 35 kDa (Fig. 2A, open arrowheads). Lysosomal membrane proteins of 51, 45, 41, and 33 and lysosomal matrix proteins of 52 and 36 kDa were less abundant in old rats (Fig. 2A, unmarked).

View larger version (84K): [in this window] [in a new window]   Fig. 2.   Comparison of the protein content of lysosomes from livers of young and old rats. A, cytosolic fractions (CYT), lysosomes (LYS), lysosomal membranes (L.MB) and lysosomal matrices (L.MTX) (100 µg of protein) isolated from livers of 3- (Y) and 22-month-old (O) rats as described under "Experimental Procedures" were subjected to SDS-PAGE and stained with Coomassie Brilliant Blue R-250. Proteins showing increased levels with age are indicated by arrowheads (filled, lysosomal membrane proteins; open, lysosomal matrix proteins). B and C, liver homogenates (HOM) and lysosomes (LYS) (100 µg of protein) resolved by electrophoresis as in A were analyzed by immunoblot for their content on lysosomal membrane (B) and lysosomal matrix (C) proteins. Lamp I, lysosome-associated membrane protein type I; Cath D and L cathepsins D and L; prec, precursor. Arrows indicate different size subunits of the mature cathepsins.

We identified by sequencing the 56-kDa band that increased with age in the lysosomal membrane to be the 56-kDa subunit of the vacuolar H⁺-ATPase. In addition, we identified by sequencing the 45-kDa band that decreased in the lysosomal membrane with age to be acid phosphatase. Other lysosomal membrane proteins such as lamp1 were detected at equivalent levels in both groups of lysosomes (Fig. 2B). In lysosomal matrices the levels of cathepsin D and L were slightly higher in lysosomes from old animals (Fig. 2C). The different molecular weight bands correspond to precursor (prec) and subunits (arrows) of cathepsins.

Age-related Changes in Chaperone-mediated Autophagy-- Lysosomes isolated from old rats showed lower degradation rates for GAPDH and RNase S-peptide than lysosomes from young rats (Fig. 3A). In both age groups the addition of an ATP-regenerating system and cytosolic hsc73 increased substrate degradation, but differences between young and old lysosomes persisted.

View larger version (55K): [in this window] [in a new window]   Fig. 3.   Changes in lysosomal chaperone-mediated autophagy activity with age. Degradation of [14C]GAPDH (A and B) and [3H]RNase S-peptide (A) by intact lysosomes from 3- (black) and 22-(gray) month-old rat livers (A) and PDL 22 (black) and 52 (gray) human fibroblasts in culture (B) (these fibroblasts stop dividing at PDL 55-65) was measured as described under "Experimental Procedures." Samples were incubated without additions (NONE) or in the presence of bovine brain hsc73 and an ATP-regenerating system (HSC/ATP). Values are the mean ± S.D. of 6-8 different experiments. Differences between age groups and PDLs are significant for p < 0.01 (*) or p < 0.001 (**). C, lysosomes isolated from 3-(black) and 22-month-old (gray) rat livers and a mixture of similar amount of lysosomes from both groups (LYS MIX; hatched) were incubated with [14C]GAPDH without additions (NONE) or in the presence of hsc73 and an ATP-regenerating system (HSC/ATP), and proteolysis was measured as in A. Values are mean ± S.D. of three different experiments. Open bars show the calculated average value of proteolysis obtained from young and old rat lysosomes separately. D, lysosomes isolated from 3- and 22-month-old rat livers and from a homogenate of similar amount (g) of livers from both groups (HOMOG MIX; hatched) were analyzed for their ability to degrade [14C]GAPDH as described in A.

We then compared chaperone-mediated autophagy in lysosomes isolated from human fibroblasts that had undergone 22 or 52 population doubling levels (PDLs). Lysosomes from senescent cells showed reduced rates of degradation of GAPDH both in the absence or presence of hsc73 and ATP (Fig. 3B). The presence of a higher content of nonlysosomal proteins in the matrix of lysosomes from old animals or senescent cells did not contribute to the decreased degradation of the substrate proteins because the same amount of lysosomal matrices from both age groups showed similar proteolytic rates (data not shown).

To determine whether or not the reduced chaperone-mediated autophagy in aging was caused by diffusible inhibitors in the lysosomal fractions isolated from old rat livers or activators in lysosomes from young rats, we mixed together lysosomes from young and old rats. Degradation of GAPDH by the mixed fraction was not significantly different from the theoretical average value of the activity of each group considered separately (Fig. 3C). Furthermore, we homogenized livers from young and old rats together and then isolated the lysosomes contained in that mixture. The rates of GAPDH degradation by those lysosomes were also exactly intermediate between the rates obtained with lysosomes from separately homogenized young and old rat livers (Fig. 3D). Consequently, diffusible inhibitors or activators were not evident in the lysosomal preparations.

We then compared the time course and saturability of chaperone-mediated autophagy of [¹⁴C]GAPDH by lysosomes from young and old rats. In contrast to the nearly linear kinetics for lysosomes from young rats (26, 31), lysosomes from old rats rapidly degraded GAPDH during the first 10 min of the incubation, but the subsequent degradation rate slowed (Fig. 4A). The K_m values for GAPDH degradation were lower in lysosomes from old rats than in lysosomes from young rats suggesting fewer substrate binding sites for chaperone-mediated autophagy in old animals (Fig. 4B). We found no differences between both age groups when we performed similar experiments with only lysosomal matrices (data not shown), suggesting that a defect in steps prior to the degradation of the substrate is responsible for the decreased chaperone-mediated autophagy rates in old rats. Despite the different K_m, both age groups show similar V_max (Fig. 4B) suggesting no differences in binding affinity between age groups although there was a decrease in the number of receptors or channels at the lysosomal membrane with age.

View larger version (47K): [in this window] [in a new window]   Fig. 4.   Changes in the kinetics and saturability of chaperone-mediated autophagy in rat liver with age. A, intact lysosomes from livers of 3- () and 22-month-old () rats were incubated with [14C]GAPDH under standard conditions and rates of protein degradation at the indicated times were determined as described in the legend to Fig. 3. Values are the mean ± S.D. of four different experiments. Differences are significant for p < 0.01 (*) or p < 0.001 (**). B, lysosomes were incubated as in A but with increasing concentrations of [14C]GAPDH for 30 min at 25 °C. At the end of the incubation, samples were processed as in A. Values are the mean of six different experiments. C, intact lysosomes isolated from young (Y, 3-month-old) and old (O, 22-month-old) rat livers were preincubated with chymostatin for 10 min at 4 °C and then incubated with RNase A (left) or GAPDH (right) (50 µg of protein) for 30 min at 37 °C. The indicated samples were treated with proteinase K, centrifuged, and subjected to SDS-PAGE and immunoblotted with specific antibodies against GAPDH or RNase A. The lower arrow indicates an intermediate transport form of RNase A as described previously (26). D, lysosomes from 3- and 22-month-old rats were incubated as in C with RNase A alone or in the presence of a similar amount of GAPDH (G) or ovalbumin (O). Samples were processed as in C. E, isolated membranes from 3-month-old (Y, young) and 22-month-old (O, old) rat liver lysosomes were incubated with RNase A (left) and GAPDH (right) for 30 min at 37 °C. At the end of the incubation, lysosomal membranes were recovered by centrifugation, washed in incubation buffer, and processed as in C. Shown are samples from two different young and old rats.

Binding and Uptake of Substrate Proteins by Lysosomes from Livers of Young and Old Rats-- We have previously developed an in vitro system that allows the separate analysis of binding and uptake of substrate proteins by lysosomes (26, 28). Binding of RNase A and GAPDH was significantly lower to the membrane of intact lysosomes isolated from livers of old rats (Fig. 4C, lanes 1 and 2). The uptake of both substrate proteins by lysosomes was also significantly lower in old rats (Fig. 4C, lanes 3 and 4). We found no age-related differences in the ability of chymostatin to inhibit the degradation of the substrate proteins by the lysosomal matrices or in the integrity of the lysosomal membrane after treatment with proteinase K (data not shown). Binding and uptake of RNase A and GAPDH by lysosomes could be inhibited in both age groups by other substrate proteins but not by nonsubstrate proteins such as ovalbumin (Fig. 4D). Rates of substrate binding and uptake are usually related (26). To determine the contribution of binding and uptake to the decreased chaperone-mediated autophagy with age, we analyzed the binding of RNase A and GAPDH to isolated lysosomal membranes from both age groups. As shown in Fig. 4E, binding of both substrates was lower to lysosomal membranes from old rats than from young rats, confirming that there is reduced binding of substrate protein with age.

To determine whether or not a defect in uptake as well as in binding occurs with age, we analyzed the amount of a transport intermediate. During RNase A transport a 2-kDa portion of the C terminus of the protein remains outside the lysosomes whereas the rest of the protein is in the lysosomal matrix (26). In the in vitro assay, we found that the percentage of partially transported RNase A, compared with bound, was higher in lysosomes from old rats than from young rats (50% of total in old versus 25% in young rats, i.e. compare lanes 1-3 with lanes 4-6 in Fig. 4D, right). Those results suggest that at least for RNase A, there is a defect in its complete transport into lysosomes from old rats in addition to the defect in binding.

Comparison of the Cytosolic hsc73 in Livers from Young and Old Rats-- Total and cytosolic contents of hsc73 were similar in young and old rat livers (Fig. 5A). Using ATP-affinity chromatography as described before (34), we obtained similar purification yields of hsc73 from the cytosol of both age groups, suggesting that the ATP binding ability of hsc73 was preserved with age. Both purified hsc73s showed similar electrophoretic characteristics in reducing SDS-PAGE (Fig. 5B). hsc73 self-associates forming multimeric complexes, and it is present in cytosol in a monomer-oligomer equilibrium (37). After native electrophoresis of the two purified hsc73s, we found similar patterns of multimerization (monomer, dimer, and trimer shown in Fig. 5C). The binding ability of hsc73 to substrate proteins and to an Escherichia coli recombinant hsc73 did not change with age (data not shown).

View larger version (49K): [in this window] [in a new window]   Fig. 5.   Comparison of cytosolic hsc73 from young and old rat livers. A, liver homogenates (HOM), cytosol (CYT), and mitochondria (MIT) (100 µg of protein) from 3-month-old (Y, young) and 22-month-old (O, old) rat livers were subjected to SDS-PAGE and immunoblotted with a specific antibody against hsc73. B, cytosolic fractions from the same rat livers were subjected to chromatography through an ATP-agarose matrix. Flow-through (FT) and eluted hsc73 (H) were then subjected to SDS-PAGE and stained with Coomassie Blue. C, hsc73 purified from the liver cytosol of both age groups were supplemented with 5 mM ADP and subjected to native electrophoresis and immunoblotted with a specific antibody against hsc73. Arrowheads indicate hsc73 monomer, dimer, and trimer complexes. D, lysosomes isolated from 3- () and 22- () month-old rat livers were incubated with [14C]GAPDH and increasing concentrations of hsc73 from young and old rat liver cytosol. Proteolysis was calculated as described in the legend to Fig. 3. Values are the mean of six different experiments. E, lysosomes isolated from 3- (black) and 22- (gray) month-old rat livers were incubated with [14C]GAPDH without additions (NONE) or in the presence of an ATP-regenerating system and hsc73 isolated from 3- (young, HSCY) and 22-month-old (old, HSCO) rat liver cytosol. Proteolysis was calculated as described in the legend to Fig. 3. Values are the mean ± S.D. of five different experiments.

hsc73 isolated from the cytosol of young and old rat livers had similar effects on the degradation of GAPDH by intact young rat lysosomes (Fig. 5D). In both groups a stimulation of degradation can be observed at low concentrations of hsc73. An inhibition of uptake occurs at higher concentrations of hsc73 because hsc73 contains two KFERQ-related motifs that make it a substrate for chaperone-mediated autophagy that will compete for binding and uptake of other substrates (23). Supplementation of old rat liver lysosomes with hsc73 isolated from young rats did not improve their reduced function (Fig. 5E). We conclude that hsc73 in livers from young and old rats are equivalent.

The Lysosomal Chaperone ly-hsc73 in Young and Old Rat Liver-- Though the total cellular content of hsc73 did not significantly change with age (Fig. 5A), the levels of hsc73 were significantly higher in lysosomes isolated from old rats than from young rats (Fig. 6A). Both membrane-associated and matrix ly-hsc73 increased with age (Fig. 6A). Immunogold localization of ly-hsc73 (Fig. 6B) revealed that the increase in the total content of ly-hsc73 in the lysosomal fraction from old rats was caused mainly by an increase in the number of lysosomes positive for hsc73 in that fraction (Fig. 6C) rather than an increase in the amount of ly-hsc73 per lysosome (Fig. 6D).

View larger version (124K): [in this window] [in a new window]   Fig. 6.   Ly-hsc73 in young and old rat livers. A, lysosomes (LYS), lysosomal membranes (L.MB), and matrices (L.MTX) (100 µg of protein) from 3-month-old (Y, young) and 22-month-old (O, old) rat livers were subjected to SDS-PAGE and immunoblotted with a specific antibody against hsc73. B, liver lysosomes from 3- (top) and 22- (bottom) month-old rats were processed for electron microscopy and immunogold labeling with a specific antibody against hsc73 and a gold-conjugated secondary antibody as described under "Experimental Procedures." Bar, 0.5 µm. Arrowheads point out lysosomes negative for hsc73 staining. C and D, the percentage of total lysosomes with or without hsc73 (hsc+ or hsc, respectively; C) and the total number of gold particles and their distribution between lysosomal membranes and matrices in lysosomes positive for hsc73 (D) are shown. Values are mean ± S.D. of three different experiments, and a total of 300 lysosomes were counted for each age group. E, lysosomes isolated from livers of 3- and 22-month-old rats were incubated in an isotonic buffer at 37 °C. At the indicated times, samples were taken and subjected to SDS-PAGE and immunoblotted with a specific antibody against hsc73.

We have previously found an increase in the number of lysosomes positive for hsc73 after prolonged starvation in rat liver (23). Under those conditions, the increase of ly-hsc73 was caused by decreased lysosomal degradation rates of hsc73 (23). The degradation rates of the hsc73 associated with old rat liver lysosomes were also significantly lower than for young rat liver lysosomes (1.12 and 0.46% per min in young and old, respectively; Fig. 6E). These results suggest that a decrease in the lysosomal degradation of hsc73 with age in a population of lysosomes normally less active for chaperone-mediated autophagy might explain the higher number of hsc73 positive lysosomes in old rats.

Reduced Levels of Receptor for Chaperone-mediated Autophagy with Age-- In contrast to the similar levels of other lysosomal membrane proteins in young and old rats (e.g. lamp1, see Fig. 2B), levels of lamp2a, a receptor for chaperone-mediated autophagy at the lysosomal membrane, were significantly lower in lysosomes from old rats (Fig. 7A, top). The lysosomal decrease was caused mainly by lower levels of lamp2a at the lysosomal membrane (Fig. 7A, top). When we substituted the antibody specific for lamp2a for an antibody against the common luminal region of all forms of lamp2, differences were not evident (Fig. 7A, middle). A similar decrease in the content of lamp2a at the lysosomal membrane was also evident when comparing early and late PDLs of human fibroblasts in culture (Fig. 7A, bottom).

View larger version (36K): [in this window] [in a new window]   Fig. 7.   Lamp2a in young and old lysosomes. A, homogenates (HOMOG), lysosomes (LYSOS), lysosomal membranes (L.MB), and matrices (L.MTX) from 3- (Y, young) and 22- (O, old) month-old rat livers and from human fibroblasts at the indicated PDLs were subjected to SDS-PAGE and immunoblotted with specific antibodies against the cytosolic tail of lamp2a, a luminal region common to all rat lamp2s (lamp2s) or to human lamp2 and lamp1 as labeled. B, lysosomes from 3- (black) and 22- (gray) month-old rats were processed for electron microscopy and immunogold labeling with a specific antibody against lamp2a and a gold-conjugated secondary antibody as described under "Experimental Procedures." The total amount and membrane and matrix distribution of gold particles in 300 lysosomes from each group was quantified. Values are mean ± S.D. of three different experiments. C, lysosomal membranes from 3-(Y, young) and 22- (O, old) month-old rat livers were normalized to equivalent amounts of lamp2a and then subjected to SDS-PAGE and immunoblotting with a specific antibody against lamp2a (lanes 1 and 2) or binding to [14C]GAPDH (lanes 3 and 4) and [14C]hsc73 (lanes 5 and 6) as described under "Experimental Procedures."

We also confirmed the decrease of lamp2a levels in lysosomes from old animals by electron microscopy and immunogold labeling (Fig. 7B). The number of gold particles for the same number of lysosomes was 30% lower in the preparations from old rat livers than from young rats. The decrease was mainly because of lower membrane levels of lamp2a because a slightly higher number of gold particles were detected in the matrices of old rat liver lysosomes.

The substrate binding ability of lamp2a is preserved with age. We adjusted the amount of lysosomal membrane proteins separated by SDS-PAGE so that the total levels of lamp2a would be the same for lysosomes isolated from young and old rats. Under those conditions, the amount of [¹⁴C]GAPDH bound was comparable for both age groups (Fig. 7C, lanes 3 and 4). This was also true for the binding of hsc73 to lamp2a (Fig. 7C, lanes 5 and 6). This result agrees with the analysis performed for binding of GAPDH to intact lysosomes (Fig. 4B). The number of binding sites declined with age, but the binding affinity was not altered.

Time Course of Age-related Changes in Chaperone-mediated Autophagy-- We then analyzed rates of chaperone-mediated autophagy and each of its different steps in 3-, 9-, and 22-month-old rats. As show in Fig. 8A, both binding and uptake of substrate proteins were progressively lower with age. However, the decrease in substrate uptake with age was much more pronounced than for substrate binding. Levels of lamp2a but not other lamp2 proteins at the lysosomal membrane gradually decreased with age at a rate comparable with the decrease detected for substrate uptake (Fig. 8B). Levels of ly-hsc73 in the group of lysosomes with low activity for chaperone-mediated autophagy in 9-month-old rats remained unchanged when compared with 3-month-old rats (Fig. 8C). We only found a significant increase in ly-hsc73 positive lysosomes in the oldest age group. These results suggest that the changes in ly-hsc73 levels with age may represent a later compensatory mechanism for decreased chaperone-mediated autophagy.

View larger version (23K): [in this window] [in a new window]   Fig. 8.   Time course of the age-related changes in chaperone-mediated autophagy. A intact lysosomes from 3-, 9-, and 22-month-old rats were incubated with GAPDH under standard conditions and lysosomal binding () and uptake () of GAPDH was analyzed by immunoblot as in Fig. 4. Immunoblots were quantified by densitometry. B, lysosomal membranes and matrices from the same age groups as in A were subjected to immunoblot with specific antibodies against lamp2a () or to all lamp2s (). The graph represents the lysosomal membrane levels of lamp2a and lamp2s. Values were obtained by densitometric quantification of three different immunoblots similar to the ones shown here. C, lysosomes with high (hsc+) and low (hsc) activity for chaperone-mediated autophagy were isolated from different aged rat livers and immunoblotted with a specific antibody against hsc73.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Chaperone-mediated autophagy in rat liver decreases with age (Fig. 3A). In this work we demonstrate that binding and uptake of substrate proteins by lysosomes (Figs. 4 and 8) are the steps affected by age. We found no age-related changes in the amount or activities of the cytosolic chaperone hsc73 (Fig. 5). The lower rates of substrate uptake by lysosomes with age (Fig. 8A) directly correlated with a decrease in the lysosomal membrane levels of lamp2a, a receptor for chaperone-mediated autophagy (Fig. 8B). Interestingly, the number of lysosomes containing hsc73 in their matrix, and therefore active for chaperone-mediated autophagy, increased at advanced age (Figs. 6, B and C and 8C). We propose here that the increase in the number of lysosomes involved in this pathway in old rats might result from a late attempt to compensate for the decreased chaperone-mediated autophagy.

The similarity of the lysosomes isolated from young and old rat livers (Fig. 1 and Tables I and II) clearly contrast with the dramatic age-related changes in the lysosomal compartment described histologically by other authors (35, 36, 38) who found an increase in the number and size of secondary lysosomes and of ceroid/lipofuscin deposits with age. The similarity of lysosomes isolated from young and old rats is probably because our lysosomal purification procedures have been optimized to obtain a lysosomal preparation mainly enriched in lysosomes active for chaperone-mediated autophagy. Thus the abnormally functioning lipofuscin-loaded lysosomes reported in other studies were not present in the lysosomal fraction analyzed here (39).

Levels of lamp2a at the lysosomal membrane are regulated by two different mechanisms, degradation of lamp2a at the lysosomal membrane and changes in the dynamic distribution of lamp2a between the membrane and the matrix (40). Whether the lower levels of lamp2a in the membrane of old rat lysosomes results from alterations of those regulatory parameters or from other completely independent processes needs to be further investigated.

Interestingly, rates of substrate protein uptake decreased more dramatically with age than did substrate binding (Fig. 8A), and a better correlation can be established between the decrease in substrate uptake and in lysosomal membrane levels of lamp2a (Fig. 8B). A possible explanation for those results is a dissociation between the binding and uptake steps with age. Thus, it is possible that under normal conditions binding of the substrate proteins to lamp2a is efficiently coupled to transport. If that coupling mechanism becomes defective with age, lower rates of protein uptake might result for the same levels of protein binding.

Other factors may contribute to the decrease in chaperone-mediated autophagy with age. Substrate proteins could undergo age-related changes that diminish their lysosomal uptake. However, we have found no evidence for substrate protein modification in GAPDH, RNase A, IB, or hsc73 with respect to their ability for binding and uptake by normal lysosomes (data not shown).

The cytosolic levels and in vitro stimulatory effect on chaperone-mediated autophagy of the cytosolic hsc73 isolated from rat livers remain unchanged with age (Fig. 5). However it is still possible that other cytosolic components with a modulatory effect on hsc73 might undergo age-related changes and modify hsc73 activity in vivo. The identification of those components will allow us address those questions in the future.

Changes in the intralysosomal environment could also contribute directly or indirectly to the decreased lysosomal activity with age. Changes in intralysosomal pH or levels of oxidative agents could result in altered function of proteins involved in the transport of substrates such as hsc73 or lamp2a. Changes in some lysosomal matrix properties with age could, for example, modify the rates of lamp2a degradation at the lysosomal membrane or block the reinsertion of the matrix lamp2a back into the lysosomal membrane (40) and consequently cause the decrease in membrane levels of lamp2a observed in old rat lysosomes.

By analogy with what we have previously described for rats subjected to prolonged starvation (26), the decreased degradation rates of hsc73 inside lysosomes not active for chaperone-mediated autophagy in old rat livers (Fig. 6E) would make them competent for substrate uptake. This seems to be an adaptation activated late in age (Fig. 8C) but the larger number of lysosomes putatively active for chaperone-mediated autophagy in the oldest rats still does not compensate for the decreased levels of receptor protein. We hypothesize that the attempted recruitment of more lysosomes for chaperone-mediated autophagy during aging might be a secondary effect of the decline in chaperone-mediated autophagy because of reduced lamp2a levels.

It is still unknown how ly-hsc73 reaches the lysosomal matrix. hsc73 contains two KFERQ-related motives, and thus part of its transport can take place by chaperone-mediated autophagy. However, the existence of a different mechanism for hsc73 transport is also possible because the protein can be efficiently transported and degraded by lysosomes with lower activity for chaperone-mediated autophagy (23). An increase in the rates of lysosomal uptake of hsc73 by that alternative mechanism with age may also explain the described higher levels of ly-hsc73.

We have previously reported a decrease in chaperone-mediated autophagy in senescent cells in culture (7, 25). Here we show a similar decrease in rat liver and demonstrate that it is mainly caused by a defect in substrate binding and uptake because of a reduced level of lamp2a. In both systems, cultured cells and rat liver, levels of lamp2a at the lysosomal membrane decrease with age and that decrease correlates with the decreased chaperone-mediated autophagy activity. These results support the use of senescent human fibroblasts for the study of aging at least with regard to the decline in chaperone-mediated autophagy.

In young cells in culture we have previously demonstrated that overexpressing lamp2a results in increased rates of chaperone-mediated autophagy (21). Using similar experimental approaches we should be able to correct the decreased levels of lamp2a with age. We can then determine whether correcting this protein degradation pathway decreases the levels of abnormal proteins that accumulate with age.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants AG00829 (to A. M. C.) and AG06116 (to J. F. D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence may be addressed: Dept. of Physiology, Tufts University School of Medicine,136 Harrison Ave, Boston, MA 02111. Tel.: 617-636-6707; Fax: 617-636-0445; E-mail: jdice@opal.tufts. edu or ana.cuervo{at}tufts.edu.

Published, JBC Papers in Press, May 9, 2000, DOI 10.1074/jbc.M002102200

	ABBREVIATIONS

The abbreviations used are: hsc73, 73-kDa heat-shock cognate protein; MOPS, 3-(N-morpholino)propanesulfonic acid; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ly-hsc73, lysosomal hsc73; lamp, lysosome-associated membrane protein; PDL, population doubling level; S-peptide, residues 1-20 of RNase A; PAGE, polyacrylamide gel electrophoresis.

	REFERENCES

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