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• W2124165909 abstract "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. 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. 73-kDa heat-shock cognate protein 3-(N-morpholino)propanesulfonic acid glyceraldehyde-3-phosphate dehydrogenase lysosomal hsc73 lysosome-associated membrane protein population doubling level residues 1–20 of RNase A polyacrylamide gel electrophoresis One of the common characteristics of senescent cells is the accumulation of abnormal proteins in the cytosol (1Makrides S. Biol. Rev. 1983; 83: 393-422Google Scholar). The described protein alterations are mostly post-translational modifications (2Gafni A. J. Am. Geriatr. Soc. 1997; 45: 871-880Crossref PubMed Scopus (33) Google Scholar) and probably result from the inevitable exposure to damaging agents such as oxygen and glucose. All cells have specific mechanisms to eliminate damaged proteins (3Dice J.F. Lysosomal Pathways of Protein Degradation. Molecular Biology Intelligence Unit, Landes Bioscience, Austin, TX2000Crossref Google Scholar, 4DeMartino G. Slaughter C. J. Biol. Chem. 1999; 274: 22123-22126Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar, 5Carafoli E. Molinari M. Biochem. Biophys. Res. Commun. 1998; 247: 193-203Crossref PubMed Scopus (340) Google Scholar). Age-related defects in intracellular proteolytic systems have been proposed to cause the accumulation of abnormal proteins (6Dice J.F. Physiol. Rev. 1993; 73: 149-159Crossref PubMed Scopus (92) Google Scholar). This hypothesis was initially supported by the fact that overall rates of protein degradation decline with age (1Makrides S. Biol. Rev. 1983; 83: 393-422Google Scholar,7Dice J.F. J. Biol. Chem. 1982; 257: 14624-14627Abstract Full Text PDF PubMed Google Scholar).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 (8Cuervo A.M. Dice J.F. Front. Biosci. 1998; 3: 25-43Crossref PubMed Google Scholar). The amount of ubiquitinated protein increases in “old” tissues, but its degradation rate does not significantly change with age (9Pan J. Short S.R. Goff S.A. Dice J.F. Exp. Gerontol. 1993; 28: 39-49Crossref PubMed Scopus (28) Google Scholar). 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 (10Shibatani T. Ward W.F. J. Gerontol. 1996; 51: B316-B322Crossref Scopus (84) Google Scholar). An increase rather than a decrease in calcium-dependent degradation of proteins with age has been reported in different tissues (11Glaser T. Schwarz-Ben Meir N. Barnoy S. Barak S. Eshhar Z. Kosower N. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7879-7883Crossref PubMed Scopus (48) Google Scholar).Proteins can be transported to lysosomes for degradation following several different pathways. The best characterized are endocytosis, crinophagy, macroautophagy, microautophagy, and chaperone-mediated autophagy (3Dice J.F. Lysosomal Pathways of Protein Degradation. Molecular Biology Intelligence Unit, Landes Bioscience, Austin, TX2000Crossref Google Scholar, 12Cuervo A.M. Dice J.F. J. Mol. Med. (Berl. ). 1998; 76: 6-12Crossref PubMed Google Scholar). 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 (7Dice J.F. J. Biol. Chem. 1982; 257: 14624-14627Abstract Full Text PDF PubMed Google Scholar, 13Terman A. Gerontology. 1995; 41: 319-326Crossref PubMed Scopus (137) Google Scholar, 14Koenig J. Edwardson J.M. Trends Pharmacol. Sci. 1997; 18: 276-287Abstract Full Text PDF PubMed Scopus (299) Google Scholar).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 (15Backer J. Bourret L. Dice J.F. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 2166-2170Crossref PubMed Scopus (51) Google Scholar, 16Wing S. Chiang H.L. Goldberg A.L. Dice J.F. Biochem. J. 1991; 275: 165-169Crossref PubMed Scopus (114) Google Scholar, 17Cuervo A.M. Knecht E. Terlecky S. Dice J.F. Am. J. Physiol. 1995; 269: C1200-C1208Crossref PubMed Google Scholar). 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 (3Dice J.F. Lysosomal Pathways of Protein Degradation. Molecular Biology Intelligence Unit, Landes Bioscience, Austin, TX2000Crossref Google Scholar, 12Cuervo A.M. Dice J.F. J. Mol. Med. (Berl. ). 1998; 76: 6-12Crossref PubMed Google Scholar). 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 (18Dice J.F. Trends Biochem. Sci. 1990; 15: 305-309Abstract Full Text PDF PubMed Scopus (520) Google Scholar). 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 (19Chiang H.-L. Terlecky S.R. Plant C.P. Dice J.F. Science. 1989; 246: 382-385Crossref PubMed Scopus (697) Google Scholar, 20Terlecky S. Dice J.F. J. Biol. Chem. 1993; 268: 23490-23495Abstract Full Text PDF PubMed Google Scholar). The chaperone-substrate protein complex binds to the lysosomal membrane through a receptor protein, the lysosome-associated membrane protein type 2a (lamp2a) (21Cuervo A.M. Dice J.F. Science. 1996; 273: 501-503Crossref PubMed Scopus (693) Google Scholar). 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 (22Agarraberes F. Terlecky S.R. Dice J.F. J. Cell Biol. 1997; 137: 825-834Crossref PubMed Scopus (248) Google Scholar, 23Cuervo A.M. Dice J.F. Knecht E. J. Biol. Chem. 1997; 272: 5606-5615Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). 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 (17Cuervo A.M. Knecht E. Terlecky S. Dice J.F. Am. J. Physiol. 1995; 269: C1200-C1208Crossref PubMed Google Scholar, 20Terlecky S. Dice J.F. J. Biol. Chem. 1993; 268: 23490-23495Abstract Full Text PDF PubMed Google Scholar). The level of the receptor at the lysosomal membrane is the rate-limiting step in protein binding and uptake (12Cuervo A.M. Dice J.F. J. Mol. Med. (Berl. ). 1998; 76: 6-12Crossref PubMed Google Scholar, 21Cuervo A.M. Dice J.F. Science. 1996; 273: 501-503Crossref PubMed Scopus (693) Google Scholar). 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 (24Jadot M. Wattiaux R. Mainferme F. Dubois F. Claessens A. Wattiaux-De Coninck S. Biochem. Biophys. Res. Commun. 1996; 223: 353-359Crossref PubMed Scopus (22) Google Scholar).We have previously reported a decrease in chaperone-mediated autophagy using confluent cultures of senescent human fibroblasts (7Dice J.F. J. Biol. Chem. 1982; 257: 14624-14627Abstract Full Text PDF PubMed Google Scholar, 25Okada A. Dice J.F. Mech. Ageing Dev. 1984; 26: 341-356Crossref PubMed Scopus (29) Google Scholar). 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.DISCUSSIONChaperone-mediated autophagy in rat liver decreases with age (Fig.3 A). 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. 8 A) directly correlated with a decrease in the lysosomal membrane levels of lamp2a, a receptor for chaperone-mediated autophagy (Fig. 8 B). 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 8 C). 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 (35De Priester W. Van Manen R. Knook D. Mech. Ageing Dev. 1984; 26: 205-216Crossref PubMed Scopus (21) Google Scholar, 36Schellens J. Cell Tissue Res. 1974; 155: 455-473Crossref PubMed Scopus (11) Google Scholar, 38Ferland G. Audet M. Tuchweber B. Mech. Ageing Dev. 1992; 64: 49-59Crossref PubMed Scopus (11) Google Scholar) 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 (39Terman A. Brunk U. Mech. Ageing Dev. 1998; 104: 277-291Crossref PubMed Scopus (115) Google Scholar).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 (40Cuervo A.M. Dice J.F. Traffic. 2000; 1: 570-583Crossref PubMed Scopus (221) Google Scholar). 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. 8 A), and a better correlation can be established between the decrease in substrate uptake and in lysosomal membrane levels of lamp2a (Fig.8 B). 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, IκB, 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 (40Cuervo A.M. Dice J.F. Traffic. 2000; 1: 570-583Crossref PubMed Scopus (221) Google Scholar) 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 (26Cuervo A.M. Terlecky S. Dice J.F. Knecht E. J. Biol. Chem. 1994; 269: 26374-26380Abstract Full Text PDF PubMed Google Scholar), the decreased degradation rates of hsc73 inside lysosomes not active for chaperone-mediated autophagy in old rat livers (Fig. 6 E) would make them competent for substrate uptake. This seems to be an adaptation activated late in age (Fig. 8 C) 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 (23Cuervo A.M. Dice J.F. Knecht E. J. Biol. Chem. 1997; 272: 5606-5615Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). 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 (7Dice J.F. J. Biol. Chem. 1982; 257: 14624-14627Abstract Full Text PDF PubMed Google Scholar, 25Okada A. Dice J.F. Mech. Ageing Dev. 1984; 26: 341-356Crossref PubMed Scopus (29) Google Scholar). 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 (21Cuervo A.M. Dice J.F. Science. 1996; 273: 501-503Crossref PubMed Scopus (693) Google Scholar). 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. One of the common characteristics of senescent cells is the accumulation of abnormal proteins in the cytosol (1Makrides S. Biol. Rev. 1983; 83: 393-422Google Scholar). The described protein alterations are mostly post-translational modifications (2Gafni A. J. Am. Geriatr. Soc. 1997; 45: 871-880Crossref PubMed Scopus (33) Google Scholar) and probably result from the inevitable exposure to damaging agents such as oxygen and glucose. All cells have specific mechanisms to eliminate damaged proteins (3Dice J.F. Lysosomal Pathways of Protein Degradation. Molecular Biology Intelligence Unit, Landes Bioscience, Austin, TX2000Crossref Google Scholar, 4DeMartino G. Slaughter C. J. Biol. Chem. 1999; 274: 22123-22126Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar, 5Carafoli E. Molinari M. Biochem. Biophys. Res. Commun. 1998; 247: 193-203Crossref PubMed Scopus (340) Google Scholar). Age-related defects in intracellular proteolytic systems have been proposed to cause the accumulation of abnormal proteins (6Dice J.F. Physiol. Rev. 1993; 73: 149-159Crossref PubMed Scopus (92) Google Scholar). This hypothesis was initially supported by the fact that overall rates of protein degradation decline with age (1Makrides S. Biol. Rev. 1983; 83: 393-422Google Scholar,7Dice J.F. J. Biol. Chem. 1982; 257: 14624-14627Abstract Full Text PDF PubMed Google Scholar). 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 (8Cuervo A.M. Dice J.F. Front. Biosci. 1998; 3: 25-43Crossref PubMed Google Scholar). The amount of ubiquitinated protein increases in “old” tissues, but its degradation rate does not significantly change with age (9Pan J. Short S.R. Goff S.A. Dice J.F. Exp. Gerontol. 1993; 28: 39-49Crossref PubMed Scopus (28) Google Scholar). 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 (10Shibatani T. Ward W.F. J. Gerontol. 1996; 51: B316-B322Crossref Scopus (84) Google Scholar). An increase rather than a decrease in calcium-dependent degradation of proteins with age has been reported in different tissues (11Glaser T. Schwarz-Ben Meir N. Barnoy S. Barak S. Eshhar Z. Kosower N. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7879-7883Crossref PubMed Scopus (48) Google Scholar). Proteins can be transported to lysosomes for degradation following several different pathways. The best characterized are endocytosis, crinophagy, macroautophagy, microautophagy, and chaperone-mediated autophagy (3Dice J.F. Lysosomal Pathways of Protein Degradation. Molecular Biology Intelligence Unit, Landes Bioscience, Austin, TX2000Crossref Google Scholar, 12Cuervo A.M. Dice J.F. J. Mol. Med. (Berl. ). 1998; 76: 6-12Crossref PubMed Google Scholar). 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 (7Dice J.F. J. Biol. Chem. 1982; 257: 14624-14627Abstract Full Text PDF PubMed Google Scholar, 13Terman A. Gerontology. 1995; 41: 319-326Crossref PubMed Scopus (137) Google Scholar, 14Koenig J. Edwardson J.M. Trends Pharmacol. Sci. 1997; 18: 276-287Abstract Full Text PDF PubMed Scopus (299) Google Scholar). 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 (15Backer J. Bourret L. Dice J.F. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 2166-2170Crossref PubMed Scopus (51) Google Scholar, 16Wing S. Chiang H.L. Goldberg A.L. Dice J.F. Biochem. J. 1991; 275: 165-169Crossref PubMed Scopus (114) Google Scholar, 17Cuervo A.M. Knecht E. Terlecky S. Dice J.F. Am. J. Physiol. 1995; 269: C1200-C1208Crossref PubMed Google Scholar). 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 (3Dice J.F. Lysosomal Pathways of Protein Degradation. Molecular Biology Intelligence Unit, Landes Bioscience, Austin, TX2000Crossref Google Scholar, 12Cuervo A.M. Dice J.F. J. Mol. Med. (Berl. ). 1998; 76: 6-12Crossref PubMed Google Scholar). 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 (18Dice J.F. Trends Biochem. Sci. 1990; 15: 305-309Abstract Full Text PDF PubMed Scopus (520) Google Scholar). 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 (19Chiang H.-L. Terlecky S.R. Plant C.P. Dice J.F. Science. 1989; 246: 382-385Crossref PubMed Scopus (697) Google Scholar, 20Terlecky S. Dice J.F. J. Biol. Chem. 1993; 268: 23490-23495Abstract Full Text PDF PubMed Google Scholar). The chaperone-substrate protein complex binds to the lysosomal membrane through a receptor protein, the lysosome-associated membrane protein type 2a (lamp2a) (21Cuervo A.M. Dice J.F. Science. 1996; 273: 501-503Crossref PubMed Scopus (693) Google Scholar). 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 (22Agarraberes F. Terlecky S.R. Dice J.F. J. Cell Biol. 1997; 137: 825-834Crossref PubMed Scopus (248) Google Scholar, 23Cuervo A.M. Dice J.F. Knecht E. J. Biol. Chem. 1997; 272: 5606-5615Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). 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 (17Cuervo A.M. Knecht E. Terlecky S. Dice J.F. Am. J. Physiol. 1995; 269: C1200-C1208Crossref PubMed Google Scholar, 20Terlecky S. Dice J.F. J. Biol. Chem. 1993; 268: 23490-23495Abstract Full Text PDF PubMed Google Scholar). The level of the receptor at the lysosomal membrane is the rate-limiting step in protein binding and uptake (12Cuervo A.M. Dice J.F. J. Mol. Med. (Berl. ). 1998; 76: 6-12Crossref PubMed Google Scholar, 21Cuervo A.M. Dice J.F. Science. 1996; 273: 501-503Crossref PubMed Scopus (693) Google Scholar). 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 (24Jadot M. Wattiaux R. Mainferme F. Dubois F. Claessens A. Wattiaux-De Coninck S. Biochem. Biophys. Res. Commun. 1996; 223: 353-359Crossref PubMed Scopus (22) Google Scholar). We have previously reported a decrease in chaperone-mediated autophagy using confluent cultures of senescent human fibroblasts (7Dice J.F. J. Biol. Chem. 1982; 257: 14624-14627Abstract Full Text PDF PubMed Google Scholar, 25Okada A. Dice J.F. Mech. Ageing Dev. 1984; 26: 341-356Crossref PubMed Scopus (29) Google Scholar). 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. DISCUSSIONChaperone-mediated autophagy in rat liver decreases with age (Fig.3 A). 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. 8 A) directly correlated with a decrease in the lysosomal membrane levels of lamp2a, a receptor for chaperone-mediated autophagy (Fig. 8 B). 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 8 C). 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 (35De Priester W. Van Manen R. Knook D. Mech. Ageing Dev. 1984; 26: 205-216Crossref PubMed Scopus (21) Google Scholar, 36Schellens J. Cell Tissue Res. 1974; 155: 455-473Crossref PubMed Scopus (11) Google Scholar, 38Ferland G. Audet M. Tuchweber B. Mech. Ageing Dev. 1992; 64: 49-59Crossref PubMed Scopus (11) Google Scholar) 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 (39Terman A. Brunk U. Mech. Ageing Dev. 1998; 104: 277-291Crossref PubMed Scopus (115) Google Scholar).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 (40Cuervo A.M. Dice J.F. Traffic. 2000; 1: 570-583Crossref PubMed Scopus (221) Google Scholar). 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. 8 A), and a better correlation can be established between the decrease in substrate uptake and in lysosomal membrane levels of lamp2a (Fig.8 B). 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, IκB, 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 (40Cuervo A.M. Dice J.F. Traffic. 2000; 1: 570-583Crossref PubMed Scopus (221) Google Scholar) 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 (26Cuervo A.M. Terlecky S. Dice J.F. Knecht E. J. Biol. Chem. 1994; 269: 26374-26380Abstract Full Text PDF PubMed Google Scholar), the decreased degradation rates of hsc73 inside lysosomes not active for chaperone-mediated autophagy in old rat livers (Fig. 6 E) would make them competent for substrate uptake. This seems to be an adaptation activated late in age (Fig. 8 C) 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 (23Cuervo A.M. Dice J.F. Knecht E. J. Biol. Chem. 1997; 272: 5606-5615Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). 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 (7Dice J.F. J. Biol. Chem. 1982; 257: 14624-14627Abstract Full Text PDF PubMed Google Scholar, 25Okada A. Dice J.F. Mech. Ageing Dev. 1984; 26: 341-356Crossref PubMed Scopus (29) Google Scholar). 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 (21Cuervo A.M. Dice J.F. Science. 1996; 273: 501-503Crossref PubMed Scopus (693) Google Scholar). 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. Chaperone-mediated autophagy in rat liver decreases with age (Fig.3 A). 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. 8 A) directly correlated with a decrease in the lysosomal membrane levels of lamp2a, a receptor for chaperone-mediated autophagy (Fig. 8 B). 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 8 C). 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 (35De Priester W. Van Manen R. Knook D. Mech. Ageing Dev. 1984; 26: 205-216Crossref PubMed Scopus (21) Google Scholar, 36Schellens J. Cell Tissue Res. 1974; 155: 455-473Crossref PubMed Scopus (11) Google Scholar, 38Ferland G. Audet M. Tuchweber B. Mech. Ageing Dev. 1992; 64: 49-59Crossref PubMed Scopus (11) Google Scholar) 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 (39Terman A. Brunk U. Mech. Ageing Dev. 1998; 104: 277-291Crossref PubMed Scopus (115) Google Scholar). 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 (40Cuervo A.M. Dice J.F. Traffic. 2000; 1: 570-583Crossref PubMed Scopus (221) Google Scholar). 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. 8 A), and a better correlation can be established between the decrease in substrate uptake and in lysosomal membrane levels of lamp2a (Fig.8 B). 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, IκB, 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 (40Cuervo A.M. Dice J.F. Traffic. 2000; 1: 570-583Crossref PubMed Scopus (221) Google Scholar) 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 (26Cuervo A.M. Terlecky S. Dice J.F. Knecht E. J. Biol. Chem. 1994; 269: 26374-26380Abstract Full Text PDF PubMed Google Scholar), the decreased degradation rates of hsc73 inside lysosomes not active for chaperone-mediated autophagy in old rat livers (Fig. 6 E) would make them competent for substrate uptake. This seems to be an adaptation activated late in age (Fig. 8 C) 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 (23Cuervo A.M. Dice J.F. Knecht E. J. Biol. Chem. 1997; 272: 5606-5615Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). 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 (7Dice J.F. J. Biol. Chem. 1982; 257: 14624-14627Abstract Full Text PDF PubMed Google Scholar, 25Okada A. Dice J.F. Mech. Ageing Dev. 1984; 26: 341-356Crossref PubMed Scopus (29) Google Scholar). 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 (21Cuervo A.M. Dice J.F. Science. 1996; 273: 501-503Crossref PubMed Scopus (693) Google Scholar). 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." @default.
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• W2124165909 title "Age-related Decline in Chaperone-mediated Autophagy" @default.
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