Impaired CMA Activity in LAMP-2A RNA-Interfered Cells.

CMA activity directly correlates with levels of LAMP-2A at the lysosomal membrane (16), but the consequences of blockage of LAMP-2A expression in CMA have never been analyzed before. The most direct way to assay CMA activity is by measuring the translocation of well characterized CMA substrates by isolated lysosomes in a standardized in vitro system (7, 8, 11, 12, 16). In contrast to fibroblasts lacking all three LAMP-2 isoforms, where changes in the density of lysosomes occur (20), we did not find differences in the density of the lysosomes normally active for CMA (enriched in the luminal chaperone) or in the recovery and enrichment of different lysosomal enzymes in this fraction (β-hexosaminidase and β-N-acetylglucosaminidase activities; data not shown). Levels of other lysosomal membrane proteins (LAMP-1, shown in Fig. 2A) and of cathepsins (cathepsin D, shown in Fig. 2A) in lysosomes from different LAMP-2A(−) cell clones were also comparable to controls. The integrity of the lysosomal membrane (measured as β-hexosaminidase latency; Fig. 2B) right after isolation and after different incubation times was similar to that of the lysosomes isolated from the control cells. Preserved lysosomal membrane integrity is an essential requirement to accurately assay CMA, because leakage of enzymes from the lumen would result in the extralysosomal degradation of the substrate. Degradation of two different well characterized CMA substrates (6, 11) by intact lysosomes (which recapitulates binding, uptake, and proteolysis of the substrate) was significantly lower in lysosomes isolated from different LAMP-2A(−) clones than in controls (Fig. 2C Left), revealing LAMP-2A as an essential component for this pathway. The decrease in CMA in each clone correlated well with the LAMP-2A levels remaining in lysosomes (Fig. 2A) and was due to diminished binding/uptake rather than altered lysosomal protease activity, because when the lysosomal membrane was disrupted (to facilitate free access of the proteases to the substrate), the differences between clones and control cells were no longer observed (Fig. 2C Right). Thus, stably blocking LAMP-2A expression in these cells was an effective method to reduce CMA activity and further analyze the cellular consequences of a failure in this form of autophagy.

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Fig. 2.

Impaired CMA in cells stably expressing RNAi for LAMP-2A. Lysosomes were isolated from WT or three LAMP-2A(−) clones of mouse fibroblasts (RNAi against three different regions of exon 8A). (A) Immunoblot of isolated lysosomes (15 μg of protein) for LAMP-2A, LAMP-1, and cathepsin D. (B) Hexosaminidase release from the isolated lysosomes. Values are expressed as percentage of the total lysosomal activity present in the extralysosomal medium and are the mean + SE of three different experiments. (C) Intact (Left) or disrupted (Right) lysosomes were incubated with radiolabeled GAPDH (Upper) or RNase A (Lower) for 30 min at 37°C. Proteolysis was calculated as the amount of initial acid-precipitable radioactivity (protein) transformed into acid-soluble radioactivity (amino acids and peptides) at the end of the incubation. Values are the mean + SE of five different experiments with triplicate samples. , P < 0.005; , P < 0.001.

Compensatory Activation of Macroautophagy in CMA-Impaired Cells.

Intracellular protein turnover is the result of a fine balance between protein synthesis and degradation by different proteolytic pathways (1–3). Rates of protein synthesis (Fig. 3A) and of degradation of short-lived proteins (Fig. 3B) (main substrate for the ubiquitin/proteasome system) were similar in the LAMP-2A(−) cells and in control cells [although in cells subjected to LAMP-2A blockage for >4 months, we have observed a significant decrease in the degradation of short-lived proteins attributable to a reduction in two of the three proteolytic activities of the 26S proteasome and to changes in the subunit composition of this protease (unpublished data)]. Because CMA contributes to the degradation of long-lived proteins, mainly during nutrient deprivation (4, 5), we expected LAMP-2A(−) cells to display reduced protein degradation under those conditions. Surprisingly, the degradation of long-lived proteins in LAMP-2A(−) cells after serum removal was not only not impaired but was even higher than in control cells (Fig. 3C). In fact, the increase in degradation after serum removal was inversely proportional to the levels of LAMP-2A remaining in each clone (Fig. 3D), and it contrasted with the decreased protein degradation (in hepatocytes and HeLa cells) (19–21) or the absence of changes (in mouse embryonic fibroblasts) (22) described in cells lacking all LAMP-2 isoforms. The increased protein degradation detected in LAMP-2A(−) cells was still taking place in lysosomes, because it was inhibited by ammonium chloride, a classical lysosomal inhibitor (Fig. 3C). Treatment with 3-methyladenine (3MA) under conditions known to inhibit macroautophagy reduced protein degradation in LAMP-2A(−) cells after serum removal, whereas, as reported in ref. 23, it did not have any effect in control cells (Fig. 3C). In fact, in agreement with previous reports (24), in control fibroblasts, activation of macroautophagy (3MA-dependent degradation) occurred only during the first 4–6 h of serum removal, decreasing progressively as starvation persists (Fig. 3E). In contrast, in the LAMP-2A(−) cells, macroautophagy activity was detected even in cells maintained in the presence of serum, and it persisted after prolonged serum removal (Fig. 3E). In the three different LAMP-2A(−) clones, 3MA-dependent degradation increased as their LAMP-2A content decreased (Fig. 3D).

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Fig. 3.

Altered protein turnover in LAMP-2A(−) cells. (A) Protein synthesis was measured in WT or three LAMP-2A(−) clones of mouse fibroblasts (c1, c2, and c3) as the incorporation of [³H]leucine (acid-soluble radioactivity) into proteins (acid-precipitable radioactivity) after 10 min of adding the radiolabeled amino acid into the culture medium. Values are expressed in dpm and are the mean + SE of three different experiments. (B) Degradation of short-lived proteins was measured in the same cells after labeling with [³H]leucine for 20 min. Proteolysis was calculated as described in Fig. 2C. Values are the mean + SE of three different experiments. (C) Degradation of long-lived proteins in the same cells was measured after labeling with [³H]leucine for 48 h and then plating the cells in medium supplemented (serum +) or not (serum −) with newborn calf serum. Where indicated, 15 mM NH₄Cl or 10 mM 3-methyladenine (3MA) was added into the incubation medium 6 h after removing the serum. Proteolysis was calculated as in Fig. 2C. Values are the mean + SE of four different experiments with triplicate samples. (D) The effect of removal of serum on the degradation of long-lived proteins was calculated in experiments carried out as described in C. Values are expressed as the ratio of proteolysis in serum − cells to that in serum + cells and are the mean + SE of five different experiments with triplicate samples. (E) The contribution of macroautophagy to total protein degradation was calculated in experiments similar to the ones described in C by adding 3MA 2 h before the measuring times. Values are expressed as percentage of lysosomal proteolysis sensitive to 3MA (NH₄Cl-sensitive) and are the mean + SE of four different experiments with triplicate samples. , P < 0.01; , P < 0.005; , P < 0.001.

This constitutive activation of macroautophagy in cells with impaired CMA was further confirmed by directly analyzing different macroautophagy markers (Fig. 4). Conversion of the microtubule-associated protein 1 light chain 3 (LC3), a cytosolic protein that conjugates to phosphatidylethanolamine in the membrane of autophagosomes, from an 18-kDa form (LC3-I) to a faster-migrating form (16 kDa; LC3-II), is a well accepted measurement of the amount of autophagic vacuoles inside cells (25). Consistent with the constitutive activation of macroautophagy, we found higher levels of LC3-II in LAMP-2A(−) cells (Fig. 4A shows immunoblots for LC3 with an antibody that recognizes the two LC3 forms and an antibody with higher affinity for the LC3-II form). As in control cells, the removal of serum in LAMP-2A(−) cells reduced the amount of LC3-II (Fig. 4A, lane 8), reflecting increased clearance of autophagic vacuoles by lysosomes under those conditions. In fact, part of the LC3-II associated to autophagosomes is normally degraded in lysosomes after these two compartments fuse, and only when this degradation is inhibited can an accurate measurement of autophagic rates be obtained (26). In the presence of protease inhibitors, levels of LC3-II in LAMP-2A(−) cells were comparable in the presence and absence of serum (lanes 11 and 12) but were always higher than in control cells (lanes 9 and 10), even when starvation was prolonged up to 24 h. These results, together with the proteolysis data (Fig. 3E), are compatible with similar rates of autophagosome formation in LAMP-2A(−) cells maintained in the presence or absence of serum but with more efficient clearance during nutrient deprivation. Changes in the intracellular distribution of LC3 also supported constitutive activation of macroautophagy in LAMP-2A(−) cells (Fig. 4B). In contrast to the diffuse cytosolic localization of LC3 in serum-supplemented control cells, LC3 was detected in LAMP-2A(−) cells as bright puncta (presumably, autophagic vacuoles). This LC3 vesicular pattern was observed in both groups of cells during the first hours of serum removal but persisted after prolonged starvation up to 24 h in LAMP-2A(−) cells only (18-h starvation is shown; Fig. 4B Bottom).

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Fig. 4.

Up-regulation of macroautophagy in cells with defective CMA. (A) (Left) Cellular extracts (150 μg of protein) of WT or LAMP-2A(−) mouse fibroblasts were immunoblotted for LC3 with a selective antibody that recognizes both LC3-I and LC3-II proteins (lanes 1–4) and an antibody that recognizes only LC3-II (lanes 5–12). Cells were maintained for 24 h in medium supplemented (+S) or not (−S) with serum. In lanes 9–12, cells were treated with pepstatin A and E-64 as described in ref. 26. Immunoblot for actin is shown at the bottom as a loading control. (Right) The increase in the levels of LC3-I and LC3-II in LAMP-2A(−) cells compared with control was calculated after densitometric quantification of four immunoblots such as those shown here. (B) Immunostaining for endogenous LC3 in WT or LAMP-2A(−) mouse fibroblasts maintained in serum-supplemented medium (serum +) or after 4 or 18 h of removing the serum from the culture medium (serum −) (similar patterns were observed up to 24 h after serum removal). Insets show particular areas at higher magnification to better visualize the ring-shaped vesicles that correspond to autophagic vacuoles. (C and D) Electron microscopy micrographs of WT and three LAMP-2A(−) clones of mouse fibroblasts (c1–c3) maintained in the absence (C) or presence (clone c2) (D) of serum for 12 h. Arrows point to autophagic vacuoles. Insets show at higher magnification structures with morphological characteristics compatible with autophagic vacuoles. (E) The number of autophagic vacuoles per field (Left) and the cytosolic area occupied by those vesicles (Right) was calculated in micrographs such as those in C and D. (F Upper) WT and LAMP-2A(−) cells were transiently transfected with the cDNA for GFP-LC3 alone or plus the cDNA for human LAMP-2A or mouse LAMP-2B, as labeled. The mean number of GFP-positive vesicles (shown as puncta) per cell was quantified in cells maintained in the presence or absence of serum. Fifteen to 30 transfected cells were counted per experiment. , P < 0.05; , P < 0.01; , P < 0.001. (F Lower) A representative picture of cells maintained with serum. Nuclei were labeled with DAPl (blue) in B and F.

We also confirmed the presence of a higher content of autophagic vacuoles in LAMP-2A(−) cells by electron microscopy (Fig. 4 C–E). The morphology of most of the autophagic vesicles observed was compatible with that of autophagolysosomes (single-membrane vesicles with partially digested material in their lumen and positive for cathepsin D immunostaining) (Fig. 4 C and D and data not shown). Removal of serum further accelerated the fusion/degradation of autophagic vacuole contents by lysosomes as their size became smaller (for the same number of vesicles, the cytosolic area occupied by the vacuoles was smaller) (Fig. 4E Right). Supporting an increased macroautophagy activity in LAMP-2A(−) cells, we also found expansion of the intracellular acid compartment (stained with monodansylcadaverine) (Fig. 7A, which is published as supporting information on the PNAS web site) and increased levels of other autophagy-related proteins such as beclin (human homologue of Atg6) and Atg12/5 conjugate complex (Fig. 7B). The constitutive activation of productive macroautophagy (involving continuous formation and clearance of autophagic vacuoles) observed in the LAMP-2A(−) cells clearly differs from the impairment in macroautophagy described when all three LAMP-2 isoforms are absent (19, 21) (autophagic vacuoles form but are not properly eliminated).

The decrease in LAMP-2A levels seems to affect autophagy but not other lysosomal functions. In LAMP-2A(−) cells, we found normal rates of internalization by both fluid-phase and receptor-mediated endocytosis (Fig. 8, which is published as supporting information on the PNAS web site) and proper lysosomal degradation of the internalized products (Fig. 8A).

We further confirmed that the observed increase in macroautophagy was directly related to the lack of LAMP-2A (and, consequently, poor CMA activity) and not just to the reduction in total levels of LAMP-2 (independent of the isoform type). Transient transfection of the LAMP-2A(−) cells with human LAMP-2A [which escapes the interference effect but is functionally interchangeable with the mouse protein (7, 16)] significantly reduced the number of LC3-positive vacuoles in the cytosol to levels similar to those observed in control cells, in both the presence and the absence of serum (Fig. 4F). In contrast, the number of autophagic vacuoles was not reduced if LAMP-2A(−) cells were transiently transfected with mouse LAMP-2B (Fig. 4F). Expression levels of both transfected proteins and their proper targeting to lysosomes were verified by immunoblot and immunofluorescence, respectively (data not shown). These results strongly support that activation of macroautophagy was due to the reduced levels of the 2A isoform and that the cytosolic and transmembrane regions of the protein, not the luminal region common to all three isoforms, are important for this function. There is always the possibility of a direct inhibitory role for LAMP-2A over macroautophagy, independent of its role in CMA. However, we consider this direct inhibitory role to be unlikely, because overexpression of LAMP-2A in different cell types does not significantly change macroautophagy activity (7, 16). This compensatory activation of macroautophagy when CMA is impaired could explain the up-regulation of macroautophagy that has been observed by us and others in familial forms of Parkinson's disease (12, 27), where mutant synucleins tightly associate to the lysosomal membrane, interfering with proper CMA function.

Chemicals.

Sources of chemicals and antibodies were as described in refs. 7, 9, and 11. We used the following antibodies: anti-cytosolic tail of LAMP-2A, -2B, and -2C [prepared in our laboratory (ref. 7 and C. Zhang and A.M.C., unpublished work)], anti-LAMP-1 (Developmental Studies Hybridoma Bank, Iowa City, IA), anti-cathepsins D and L (Santa Cruz Biotechnology), anti-GM130, anti-BiP and anti-caveolin-1 (BD Transduction Laboratories), anti-actin (Abcam, Cambridge, MA), and anti-beclin (Novus Biologicals, Littleton, CO). The two polyclonal antibodies against LC3, the one against Atg12, and the cDNA that codes LC3 fused to GFP were a generous gift from Noburo Mizushima (University of Tokyo, Tokyo). The cDNAs that code for human LAMP-2A and mouse LAMP-2B were obtained from the American Type Culture Collection expressed sequence tag library and inserted into the PCR3.1 mammalian expression vector (Invitrogen).

RNAi.

Stable RNAi was carried out as described in ref. 29. The sequences of the regions targeted by the siRNA in the exon 8a of the LAMP-2A gene were 5′-GACTGCAGTGCAGATGAAG-3′, 5′-CTGCAATCTGATTGATTA-3′, and 5′-TAAACACTGCTTGACCACC-3′, corresponding to bases 1198–1216, 1331–1359, and 1678–1700. The hairpin (sense–loop–antisense) for these sequences was inserted in the multicloning region of the pSuper vector (Ambion, Austin, TX). Cultured cells were transfected by the calcium phosphate method and selected for stable transfectants by resistance to Geneticine (7).

Isolation of Subcellular Fractions.

Lysosomes from cultured cells were isolated from a light mitochondrial–lysosomal fraction in a discontinuous metrizamide/Percoll density gradient as described in ref. 30. Preparations with >10% broken lysosomes, measured as β-hexosaminidase latency, were discarded.

Uptake and Degradation of Substrate Proteins by Isolated Lysosomes.

[¹⁴C]GAPDH was incubated in Mops buffer [10 mM 3-(N-morpholino)propanesulfonic acid, pH 7.3/0.3 M sucrose/1 mM DTT] with lysosomes for 30 min. Degradation of [¹⁴C]GAPDH was measured after precipitation with trichloroacetic acid as described in refs. 7, 11, and 12. Proteolysis was expressed as the percentage of the initial acid-insoluble radioactivity (protein) transformed into acid-soluble radioactivity (amino acids and small peptides) at the end of the incubation.

Intracellular Protein Turnover.

Rates of protein synthesis and degradation were measured as described in refs. 7 and 12 (see Supporting Methods, which is published as supporting information on the PNAS web site).

Fluorescence and Immunocytochemical Staining.

Immunofluorescence analysis of 3T3 cells was performed as described in ref. 7 (see Supporting Methods for the specific conditions for each type of staining). Images were acquired with an Axiovert 200 fluorescence microscope (Zeiss), subjected to deconvolution with the manufacturer's software, and prepared by using photoshop 6.0 (Adobe Systems, San Jose, CA). Quantification was carried out by using nih image j in three different Z-stack sections for each picture after thresholding.

mRNA Quantification.

mRNA quantification was carried out by using real-time PCR (see Supporting Methods).

Electron Microscopy.

Cells maintained in the presence or absence of serum were washed and fixed in 2.5% glutaraldehyde in SC (100 mM sodium cacodylate, pH 7.43) at room temperature for 45 min. The pellet was then rinsed in SC, postfixed in 1% osmium tetroxide in SC and then 1% uranyl acetate, dehydrated through a graded series of ethanols, and embedded in LX112 resin (Ladd Research Industries, Burlington, VT). Ultrathin sections were cut on a Reichert Ultracut E, stained with uranyl acetate and then lead citrate, and viewed on a JEOL 1200EX transmission electron microscope at 80 kV. Morphometric measurements were carried out at 12 h of serum removal to avoid overestimation of size of the autophagic compartment due to cell shrinkage observed after 18 h of serum removal.

Viability and Apoptosis Measurement.

Cell viability was determined by the tetrazolium salt method. The percentage of apoptotic cells was determined by FACS analysis after staining for annexin V and TUNEL by using commercially available kits. (See Supporting Methods for details.)

We thank Ms. Irene Puga for help with the FACS analysis, the Analytical Imaging Facility (Albert Einstein College of Medicine) for help with electron microscopy, and Dr. Fernando Macian and the members of our laboratory for their valuable suggestions and comments. This work was supported by National Institutes of Health/National Institute on Aging (NIH/NIA) Grant AG021904 and an Ellison Medical Foundation Research Award (to A.M.C.). A.C.M. is supported by NIH/NIA Training Grant T32AG023475. R.K. is an NIH/NIA Postbachelor Fellow.