Impaired Degradation of Mutant α-Synuclein by Chaperone-Mediated Autophagy

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Science  27 Aug 2004:
Vol. 305, Issue 5688, pp. 1292-1295
DOI: 10.1126/science.1101738


Aberrant α-synuclein degradation is implicated in Parkinson's disease pathogenesis because the protein accumulates in the Lewy inclusion bodies associated with the disease. Little is known, however, about the pathways by which wild-type α-synuclein is normally degraded. We found that wild-type α-synuclein was selectively translocated into lysosomes for degradation by the chaperone-mediated autophagy pathway. The pathogenic A53T and A30P α-synuclein mutants bound to the receptor for this pathway on the lysosomal membrane, but appeared to act as uptake blockers, inhibiting both their own degradation and that of other substrates. These findings may underlie the toxic gain-of-function by the mutants.

A30P and A53T mutations of α-synuclein, a cytosolic protein that normally exerts a presynaptic function (1), cause familial forms of Parkinson's disease (PD) (2). Because familial PD mutations in parkin and the ubiquitin carboxy-terminal hydrolase L1 (UCHL1) genes affect the ubiquitin-dependent proteasome proteolytic system, and mutations in the DJ-1 gene (PARK7 gene on chromosome 1p36) are associated with the closely related sumoylation pathway, proteasomal degradation appears to be involved at least in some PD pathogenic pathways (3). Initial reports that α-synuclein is degraded through the proteasome (4, 5) led to the idea that abnormalities in proteasomal degradation of α-synuclein underlie PD (6). Some subsequent studies failed to show alteration of α-synuclein levels by proteasomal inhibition (79), suggesting that there are alternate forms of α-synuclein degradation. Whereas proteins with short half-lives are mostly broken down by the proteasome, most cytosolic proteins with long half-lives (>10 hours) are degraded by autophagic pathways within lysosomes (1012). Lysosomal inhibitors increase intracellular levels of α-synuclein (1315), suggesting that α-synuclein may also be degraded by autophagy. Experimental overexpression of mutant α-synuclein activates macroautophagy, a form of autophagy in which large regions of cytosol are engulfed and trafficked to lysosomes (12). Although activation of macroautophagy degrades the mutant proteins (13, 16) and mislocalizes synucleins to autophagic organelles (17), inhibition of macroautophagy does not appear to alter the degradation of wild-type α-synuclein (15).

In contrast to macroautophagy, a highly specific subset of cytosolic proteins with a motif recognized by the hsc70 chaperone are selectively degraded in lysosomes by a process known as chaperone-mediated autophagy (CMA) (12, 18). Following binding of the chaperone-substrate complex to a lysosomal membrane receptor, lamp2a (19), CMA substrate proteins are translocated into the lumen for degradation by hydrolases (18, 20).

We noted that the α-synuclein sequence contains a pentapeptide sequence (95VKKDQ99) that is consistent with a CMA recognition motif (21). In rat ventral midbrain cultures that contain dopaminergic neurons maintained in serum-free medium, we confirmed that the endogenous wild-type α-synuclein exhibited a relatively long half-life (16.8 ± 2 hours; Fig. 1A) (22). In contrast to the relatively small effect of epoxomicin, a selective proteasome inhibitor, on the half-life of α-synuclein (a 2.3-hours increase in half-life; Fig. 1A), ammonium chloride, which inhibits lysosomal proteolysis independently of the form of autophagy that delivers substrates to lysosomes, strongly inhibited α-synuclein degradation (9.6-hours increase in half-life; Fig. 1A). As described previously in PC12 cells for human wild-type α-synuclein (13), addition of 3-methyladenine, an inhibitor of macroautophagy, did not modify the degradation of rat α-synuclein (16.1± 2.4 hours). It thus appears that endogenous rat α-synuclein in ventral midbrain neuronal cultures is degraded in lysosomes but not by macroautophagy. We then examined the degradation of human wild-type α-synuclein expressed in PC12 cells (16), in which serum removal activates both macroautophagy and CMA (Fig. 1B). Serum removal markedly enhanced human α-synuclein proteolysis (from a half-life of 33.1 ± 6.3 hours to 19.7 ± 2.1 hours; n =5), whereas ammonium chloride inhibited its degradation (half-life of 48.9 ± 5.4 hours and 80.3 ± 16.6 hours, in the presence or absence of serum, respectively; n =5) (Fig. 1B) (supporting online text 1).

Fig. 1.

Degradation of α-synuclein in lysosomes by CMA. (A) Effect of 15 mM NH4Cl, 10 mM 3-methyl adenine (3-MA), or 10 nM epoxomicin (Epoxo) on the degradation of α-synuclein in 35S-labeled rat ventral midbrain neuron cultures (22). The effects of these compounds on total rates of protein degradation in these cells are reported in the supplemental data (fig. S1A). (B) α-Synuclein immunoprecipitated from clonal PC12 cells stably expressing wild-type human α-synuclein (16) labeled as in (A) and maintained with (serum +) or without serum (serum –) and 15 mM NH4Cl. Immunoprecipitation controls are shown in fig. S1B. (C) Association of increasing concentrations of α-synuclein with isolated lysosomes untreated (Binding) or previously treated with proteinase inhibitors (Binding + Uptake) (19, 20, 23, 24). (D) Effect of a 2 M excess of glyceraldehyde-3-phosphate-dehydrogenase (GAPDH), ribonuclease A (RNase A), or ovalbumin (Ovalb) (23) in the lysosomal binding and uptake of α-synuclein analyzed by immunoblot as in (C). Percentages of inhibition are the mean + SE from four experiments. Inset shows a representative immunoblot for α-synuclein binding to lysosomes. (E) Effect of adding α-synuclein (WTsyn) or ovalbumin (Ovalb) at the indicated molar ratio with [14C]GAPDH on the degradation of [14C]GAPDH by intact lysosomes. Values are the mean + SE of four experiments (*P < 0.05; **P < 0.01). (F) Effect of antibody blockade of the cytosolic tail of lamp2a on the binding of α-synuclein to isolated lysosomes.

The presence of a CMA motif, however, does not guarantee that a protein is degraded by this pathway (21). The most direct test of whether a protein is a CMA substrate is to determine its binding, uptake, and degradation in isolated intact lysosomes (19, 20, 23, 24). Because synuclein protofibrils have been suggested to destabilize the membranes of synthetic vesicles (25), we first confirmed that isolated lysosomes were not disrupted by wild-type or mutant α-synuclein proteins at concentrations as high as 70 μM (fig. S4B) (22). Under these conditions, we found that purified α-synuclein added to the incubation medium was translocated into and degraded by intact lysosomes, because lysosomal protease inhibitors increased levels of lysosomal-associated α-synuclein (Fig. 1C), and much of the α-synuclein associated to lysosomes was resistant to exogenously added proteases, demonstrating protection by the lysosomal membrane (fig. S4C) (22). The established CMA substrates glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ribonuclease A (RNase A) (20, 23) both inhibited lysosomal binding and uptake of α-synuclein into intact lysosomes, whereas ovalbumin, a protein which is not a CMA substrate, had no effect on α-synuclein binding or uptake (Fig. 1D). Consistent with a CMA substrate action, α-synuclein inhibited binding and uptake of GAPDH into isolated lysosomes (fig. S4D and Fig. 1F) but had no effect on GAPDH degradation by free lysosomal enzymes (see below); the differential inhibitory effects of different CMA substrates on binding and uptake may be related to differences in affinity for the binding and translocation components in the lysosomal membrane or to different unfolding requirements (26). As previously shown for other CMA substrates, blockade of the cytosolic tail of the lysosomal receptor using a selective antibody decreased the association of α-synuclein with lysosomes (Fig. 1F) (19). Finally, we mutated the sequence 95VKKDQ99, consistent with a CMA recognition motif (21), by replacing DQ with AA, and found that, although the mutant ΔDQ and wild-type protein were similarly susceptible to degradation by lysosomal enzymes (fig. S4A), the association of the ΔDQ protein with the lysosomal membrane and its translocation into the lysosomal lumen was dramatically reduced (Fig. 2, A and B). Accordingly, GAPDH did not modify lysosomal binding of ΔDQ α-synuclein (Fig. 2A), and mutant ΔDQ did not interfere with GAPDH binding (Fig. 2C). The mutant ΔDQ protein, in contrast to the wild type, did not bind the CMA receptor, lamp2a, at the lysosomal membrane (Fig. 2D). Thus, wild-type α-synuclein is internalized and degraded in lysosomes by CMA.

Fig. 2.

A CMA targeting motif in α-synuclein. (A) Association of wild-type and mutant ΔDQ α-synuclein with isolated lysosomes untreated (Binding) or previously treated with proteinase inhibitors (binding + uptake; B+U). (Right) Effect of a 2 M excess of glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) on the association of wild-type and mutant α-synuclein to lysosomes (lanes 5 to 8). GAPDH inhibited 53.3 ± 3.2% and 42.8 ± 4.6% of wild-type α-synuclein binding and uptake, respectively, but did not have a significant effect on mutant α-synuclein binding or uptake. Values are the mean ± SE of six experiments. Uptake was calculated as in Fig. 1C. (B) Binding of wild-type and ΔDQ mutant α-synuclein to intact lysosomes at 0°C. Values are the mean ± SE from three different experiments. Inset shows a representative immunoblot with duplicate samples. (C) Binding of GAPDH to intact lysosomes alone (none) or in the presence of a 4 M excess of wild-type (WT) or mutant α-synuclein (ΔDQ). Values are the mean ± SE of three different experiments [(*) differences compared to WT; (+) differences comparing with and without GAPDH). (D) Immunoblot of lamp2a coimmunoprecipitated with wild-type and mutant α-synuclein incubated with intact lysosomes (*P < 0.05; **P < 0.01).

We then compared CMA of the pathogenic mutant and wild-type α-synuclein proteins in isolated lysosomes. Both A30P and A53T mutant α-synucleins bound intact lysosomes more strongly than did the wild-type protein, but the mutants were poorly internalized (Fig. 3A). The preferential binding of the mutant forms to this site was not simply a consequence of impaired uptake, because it was retained under lower temperature conditions in which the accumulation of substrates by CMA into the lysosomal lumen was blocked (Fig. 3B) (23). In cells expressing similar levels of wild-type and mutant α-synucleins, the amount of lamp2a that coimmunoprecipitated with mutant α-synucleins was higher than coimmunoprecipitated with the wild type (Fig. 3C; by twofold without serum and fourto sixfold with serum), confirming that the mutant proteins were tightly bound to the CMA receptor. As expected from their strong binding to the lysosomal receptor, the mutant α-synucleins inhibited GAPDH degradation in intact lysosomes more effectively than did the wild type (Fig. 3D). This inhibition was not due to an inhibition of proteolysis, because even a fivefold excess of all three forms of α-synuclein did not block degradation of GAPDH by free lysosomal proteases (Fig. 3E). Thus, the mutant forms of α-synuclein bound strongly to the CMA receptor on lysosomes but were not translocated into the lysosomal lumen, also impairing the degradation of other CMA substrate proteins.

Fig. 3.

Altered CMA of pathogenic α-synuclein mutants. (A) Binding and uptake of wild-type and A30P and A53T mutant α-synuclein by isolated lysosomes were measured as in Fig. 1C. Values are the mean ± SE of six different experiments. Inset shows a representative immunoblot. (B) Binding of wild-type and mutant α-synuclein to intact lysosomes at 0°C. Values are the mean ± SE from three different experiments. (C) Lamp2a coimmunoprecipitated with wild-type and mutant α-synucleins in the expressing PC12 cells maintained with (S+) or without (S–) serum. Values are the mean ± SE of three different experiments. Inset shows a representative immunoblot. (D) Effect of increasing concentrations of wild-type and mutant α-synucleins on the degradation of [14C]GAPDH by isolated lysosomes. Values are the mean ± SE of three different experiments. Median inhibitory concentrations (IC50) and inhibition constants (Ki) for each protein are shown. (E) Effect of a 5 M excess of wild-type or mutant α-synucleins on the degradation of [14C]GAPDH by disrupted lysosomes. Values are the mean ± SE of two different experiments (*P < 0.05; **P < 0.01).

Mutant α-synucleins were more stable than was the wild-type protein in PC12 cell clonal lines (Fig. 4A), and cells that expressed A53T or A30P mutant α-synuclein showed impaired autophagic degradation of proteins with a long half-life (Fig. 4B) (22). As in fibroblasts (27), once PC12 cells reached confluence, only a small fraction (5%) of total lysosomal protein degradation (sensitive to ammonium chloride) took place by macroautophagy (sensitive to 3-methyladenine) (Fig. 4C), and the same was true in PC12 cells that overexpressed wild-type α-synuclein. In contrast, PC12 clones overexpressing pathogenic α-synuclein mutants exhibited slower rates of long-lived protein degradation, and the remaining lysosomal protein degradation was completely blocked by 3-methyladenine, consistent with a blockade of CMA and compensatory activation of macroautophagy. These results could explain the degradation by macroautophagy of mutant, but not wild-type, α-synuclein proteins (13) (supporting online text 2). The induction of macroautophagy following blockade of normal CMA by mutant α-synucleins appears consistent with observations in cultured fibroblasts, in which blockade of CMA leads to compensatory activation of macroautophagy (28), as well as with the induction of neuronal macroautophagy by a number of stress paradigms, including the overexpression of the mutant α-synucleins (16, 29).

Fig. 4.

Impaired CMA in cells expressing pathogenic α-synuclein mutants. (A) (Top) Degradation of wild-type (WT), A53T, and A30P human α-synuclein in stably transfected clonal PC12 cells was analyzed as in Fig. 1B. (Bottom) Half-lives of α-synucleins (from three experiments) calculated from measurements at 0, 12, 24, 36, and 48 hours [(*) differences compared to wild type; (+) differences comparing serum + to serum –; *P < 0.05, **P < 0.01, +++P < 0.001]. (B) Degradation rates of long-lived proteins in serum-deprived PC12 cells (none) or PC12 cells stably expressing wild-type (WT), and mutant α-synucleins maintained without (top) or with 15 mM NH4Cl (bottom). Values are the mean ± SE of three samples in two different experiments. (C) Inhibition of degradation of long-lived proteins in the cells described in (B) by 15 mM NH4Cl or 10 mM 3-methyladenine (3-MA).

Thus, wild-type α-synuclein is efficiently degraded in lysosomes by CMA, but the pathogenic α-synuclein mutations are poorly degraded by CMA despite a high affinity for the CMA receptor. Mutant α-synucleins blocked the lysosomal uptake and degradation of other CMA substrates. CMA blockade then results in a compensatory activation of macroautophagy which, under these conditions, cannot maintain normal rates of protein degradation. Impaired CMA of pathogenic α-synuclein may favor toxic gains-of-functions by contributing to its aggregation or additional modifications, such as nitrated or dopamine-adduct formation that could further underlie PD and other synucleinopathies (3). Mutant α-synuclein also inhibits the degradation of other long-lived cytosolic proteins by CMA, which may further contribute to cellular stress, perhaps causing the cell to rely on alternate degradation pathways or to aggregate damaged proteins.

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