Ubiquitin ligase Nedd4 promotes α-synuclein degradation by the endosomal–lysosomal pathway

Abstract

α-Synuclein is an abundant brain protein that binds to lipid membranes and is involved in the recycling of presynaptic vesicles. In Parkinson disease, α-synuclein accumulates in intraneuronal inclusions often containing ubiquitin chains. Here we show that the ubiquitin ligase Nedd4, which functions in the endosomal–lysosomal pathway, robustly ubiquitinates α-synuclein, unlike ligases previously implicated in its degradation. Purified Nedd4 recognizes the carboxyl terminus of α-synuclein (residues 120–133) and attaches K63-linked ubiquitin chains. In human cells, Nedd4 overexpression enhances α-synuclein ubiquitination and clearance by a lysosomal process requiring components of the endosomal-sorting complex required for transport. Conversely, Nedd4 down-regulation increases α-synuclein content. In yeast, disruption of the Nedd4 ortholog Rsp5p decreases α-synuclein degradation and enhances inclusion formation and α-synuclein toxicity. In human brains, Nedd4 is present in pigmented neurons and is expressed especially strongly in neurons containing Lewy bodies. Thus, ubiquitination by Nedd4 targets α-synuclein to the endosomal–lysosomal pathway and, by reducing α-synuclein content, may help protect against the pathogenesis of Parkinson disease and other α-synucleinopathies.

Parkinson disease (PD) is a common neurodegenerative condition characterized primarily by motor symptoms (tremor, rigidity, bradykinesia, and postural instability) and often associated with dementia. The main pathological features of PD are the death of dopaminergic neurons and diffuse accumulation of α-synuclein in intraneuronal aggregates known as Lewy bodies and Lewy neurites (1, 2). Studies of familial cases of PD with α-synuclein gene multiplications and overexpression of α-synuclein in rodent brains indicate that the level of α-synuclein in neurons is critical in determining the onset of neurodegeneration (1, 3). Given that no consistent alterations in α-synuclein mRNA levels have been found in sporadic PD (4), impaired α-synuclein degradation is likely to contribute to the accumulation of this protein in disease states. Understanding how α-synuclein levels are regulated requires the identification of enzymes that catalyze its degradation. The mechanisms of α-synuclein degradation have been controversial. Multiple proteolytic systems, including proteasomes (5, 6) macroautophagy (7), and chaperone-mediated autophagy (8), have been implicated in α-synuclein degradation, and several ubiquitin ligases (9–12) have been reported to influence α-synuclein levels. However, most of these studies concerned overexpressed rather than endogenous α-synuclein or E3s, and direct interactions between the purified ligases and α-synuclein have not been rigorously demonstrated (9–12).

Most cell proteins are selectively targeted for degradation after conjugation to a ubiquitin chain (13). This modification involves activation of ubiquitin by the enzyme E1, transfer of the reactive ubiquitin to the ubiquitin-conjugating enzyme E2, and then conjugation by ubiquitin ligase E3 to a protein substrate or a preceding ubiquitin to form a ubiquitin chain. Ubiquitin contains seven lysines, each of which can be linked to another ubiquitin molecule through an isopeptide bond (14). Although formation of ubiquitin chains, in which the ubiquitins are covalently linked through their K48 or K11 residues, leads to the degradation of cytosolic proteins by 26S proteasomes, attachment of chains linked through K63 residues to membrane-associated proteins targets them for lysosomal degradation (14). Because a fraction of the α-synuclein in Lewy bodies is ubiquitinated (15), ubiquitin-dependent degradation may regulate the α-synuclein content in cells and could contribute to the pathogenesis of PD. Nedd4 (neuronal precursor cell-expressed, developmentally down-regulated gene 4) is a HECT-domain E3 that functions at the plasma membrane in the turnover of a number of membrane-associated proteins (16). Nedd4 contains substrate interacting (WW) domains with affinity for proline-rich motifs (16, 17), one of which we found to be present in α-synuclein (see below). Because of this role and mode of interaction with substrates, we hypothesized that Nedd4 might function as an E3 for α-synuclein.

Results

Purified Nedd4 Ubiquitinates α-Synuclein by Recognizing Its C Terminus.

To test whether Nedd4 is able to ubiquitinate α-synuclein, we incubated purified recombinant human α-synuclein with recombinant human Nedd4, E1, the E2 UbcH5, ubiquitin, and ATP. For comparison, we also assayed three purified recombinant E3s previously implicated in α-synuclein degradation—CHIP (10), SIAH2 (11), and E6-AP (12)—and Parkin, the RING-domain E3 mutated in autosomal-recessive PD and reported to ubiquitinate a glycosylated species of α-synuclein (9), as well as MuRF1, a muscle atrophy-specific E3. Only Nedd4 caused significant ubiquitination of purified α-synuclein (Fig. 1 A and B). Ubiquitination of α-synuclein was not observed in the absence of this E3 or any of its cofactors (Fig. 1). Although they failed to ubiquitinate α-synuclein, all of these other E3s had enzymatic activity, as demonstrated by their ability to ubiquitinate themselves and the universal E3 substrate, S5a (18). We also tested several other members of the WW HECT-domain family of E3s that are related to Nedd4 and expressed in brain, Nedd4-2, Smurf1, and Smurf2 (SI Appendix, Fig. S1), but found that, unlike Nedd4, none could ubiquitinate α-synuclein (Fig. 1C).

Fig. 1.

Nedd4 ubiquitinates full-length α-synuclein. (A) Recombinant α-synuclein is ubiquitinated by purified recombinant Nedd4 in the presence of E1, ATP, and UbcH5. Both oligo-ubiquitinated and poly-ubiquitinated species were seen using two anti–α-synuclein antibodies (LB509 and Syn-1). (B) Parkin, CHIP, and SHIA2, which were previously implicated in Parkinson disease, do not ubiquitinate α-synuclein. (C) Nedd4, but not the other major WW domain/HECT-domain E3s, Nedd4-2, Smurf1, and Smurf2, ubiquitinate α-synuclein. (D) Truncation of the carboxyl-terminus (aa 120–140), but not the N terminus (aa 1–40), abolishes ubiquitination of α-synuclein by Nedd4. (E) Both UbcH5 (Upper) and UbcH7 (Lower) with Nedd4 form almost exclusively K63 Ub-chain on α-synuclein. (F) Lysine residues at position 21 and 96 on α-synuclein are the major ubiquitination sites.

The Nedd4 family of enzymes specifically binds substrates containing proline-rich (PxY, PS) motifs (16, 17). The carboxyl terminus of α-synuclein contains a sequence, PVDPDNEAYEMPSEEGYQDYEPEA, of this general type. To test whether Nedd4 recognizes this region of α-synuclein, we generated recombinant α-synuclein fragments lacking either the N-terminal residues (1–40) or the C-terminal residues (120–140). Deletion of the proline-rich C terminus, but not the N terminus, of α-synuclein prevented α-synuclein ubiquitination by Nedd4 (Fig. 1D). In additional experiments, α-synuclein deletion mutants lacking 136–140 or 134–140 (kindly provided by J. Johnston, Elan Corporation) were ubiquitinated in a similar fashion as the full-length protein (SI Appendix, Fig. S2). The prevention of ubiquitin conjugation on deletion of 120–140 is not due to the loss of a critical ubiquitination site, because the C terminus of α-synuclein does not include any lysines (see below). Therefore, ubiquitination of α-synuclein by Nedd4 requires residues 120–133 (PDNEAYEMPSEEGY), a proline-rich sequence containing a PS motif.

Nedd4 Forms K63 Chains on Specific Lysine Residues of α-Synuclein.

The nature of the isopeptide linkages in the polyubiquitin chain on a protein determines the site of degradation in the cell. To learn the type(s) of linkages formed by Nedd4 on α-synuclein, we initially compared the incorporation of seven different mutant ubiquitins, which contained only one lysine residue (with the six other lysines replaced by arginines), along with seven ubiquitin mutants that contain a single lysine-to-arginine substitution (18). Both approaches indicated that Nedd4 (with either UbcH5 or UbcH7) could monoubiquitinate α-synuclein with nearly all of the ubiquitin mutants, but the formation of long polyubiquitin chains specifically required lysine 63 (SI Appendix, Fig. S3). Previous observations of the chains formed by Nedd4 on other substrates also indicated formation of K63 chains (18). We confirmed the type of chains formed on α-synuclein and identified the site of ubiquitination by mass spectrometry. Immobilized Nedd4 was incubated with ubiquitin, ATP, α-synuclein, and UbcH5 or UbcH7 as the E2 for 30 or 120 min, and after centrifugation to remove the Nedd4, the supernatant proteins were analyzed. AQUA analysis indicated that regardless of the reaction time, the E2 used, and the length of the ubiquitin chain formed, Nedd4 synthesized K63 linkages almost exclusively on α-synuclein (Fig. 1E), and that the major sites of ubiquitination on this substrate were lysines-21 and -96 (Fig. 1F).

Nedd4 Binds to α-Synuclein in Brain and Cell Extracts.

We then tested whether Nedd4 has sufficient affinity for α-synuclein in brain homogenates to be a relevant E3 in vivo. We incubated GST-Nedd4 or its yeast ortholog GST-Rsp5p with a dilute mouse brain lysate and found that endogenous mouse α-synuclein became associated with both E3s, as shown by pull-down experiments and immunoblot analysis (SI Appendix, Fig. S4). In control experiments, GST alone and GST-MuRF1 did not bind α-synuclein in the brain lysate. To test whether these proteins interact in cells, we expressed either T7-tagged Nedd4 or an empty vector and untagged human α-synuclein in HEK cells. α-Synuclein could be immunoprecipitated from lysates of cells expressing Nedd4 with anti-T7 tagged antibodies, but not from controls expressing the empty vector (Fig. 2A). Finally, to confirm that this interaction is also seen with endogenous proteins, we immunoprecipitated endogenous Nedd4 from mouse and human brain and found that α-synuclein coprecipitated with this E3 (Fig. 2B).

Fig. 2.

Nedd4 binds to α-synuclein and promotes its degradation in mammalian cells. (A) HEK cells expressing untagged α-synuclein and T7-tagged Nedd4 or empty vector were subjected to immunoprecipitation with anti-T7 antibodies. α-Synuclein is precipitated with Nedd4 from these cell lysates. (B) Anti-Nedd4 antibodies, but not control IgG, immunoprecipitates endogenous Nedd4 with α-synuclein in mouse and human brain lysates. (C) Expression of Nedd4, but not empty vector, in SH-SY5Y cells led to rapid degradation of endogenous α-synuclein measured in the presence of cycloheximide (20 μg/mL). Disappearance of α-synuclein was quantified as the ratio of intensities of the α-synuclein band to the GAPDH band. (Right) Averages of three experiments. (D) In cells transfected with Nedd4 siRNA, the basal levels of endogenous α-synuclein were increased compared with cells transfected with scrambled (scr) siRNA. (Left) A typical experiment in HEK cells. (Right) The mean content of α-synuclein was quantified as the ratio of α-synuclein to actin band intensity and shown as % control (*P < 0.01 by t test; n = 6).

Nedd4 Promotes the Degradation of Endogenous α-Synuclein by Lysosomes.

To determine whether Nedd4 influences α-synuclein levels in mammalian cells, we tested whether Nedd4 promotes the degradation of endogenous α-synuclein in SH-SY5Y cells, a human dopaminergic neuroblastoma cell line. Cells overexpressing Nedd4 or controls were treated with cycloheximide to block protein synthesis, and the drop in endogenous α-synuclein level over time was assayed. Although transient Nedd4 overexpression did not significantly alter the basal level of α-synuclein (Fig. 2C, time 0), after addition of cycloheximide, Nedd4 caused a 50% reduction in the level of α-synuclein at 8 h (when no cytotoxicity was evident). In contrast, no significant decrease in α-synuclein content was detected in cycloheximide-treated control cells at this time (Fig. 2C). Conversely, when endogenous Nedd4 content was reduced by transfection of HEK293 or SH-SY5Y cells with RNAi (Fig. 2D and SI Appendix, Fig. S5) for 48 h, the cellular content of α-synuclein increased significantly. This indicates that Nedd4 has the capacity not only to accelerate the intracellular degradation of endogenous α-synuclein, but also, at physiological levels, to decrease the cellular content of α-synuclein.

To determine whether Nedd4 targets α-synuclein for degradation by proteasomes or lysosomes, we incubated control SH-SY5Y cells or cells overexpressing Nedd4 with selective inhibitors of each degradative system, and then assessed the changes in endogenous α-synuclein levels. Proteasomal function was specifically inhibited using bortezomib (Velcade), lysosomal function with chloroquine (which prevents lysosomal acidification), and autophagy with 3-methyladenine (3MA), which blocks autophagic vacuole formation, using concentrations that maximally inhibit these systems without causing toxicity (19). Within 6 h of addition of all three inhibitors, α-synuclein content in cells was increased by approximately twofold. Therefore, endogenous α-synuclein turns over in these cells with a relatively short half-life, and proteasomes, macroautophagy, and perhaps another lysosomal process all contribute to its basal degradation (Fig. 3A). This finding of multiple routes for α-synuclein degradation provides support for the several seemingly contradictory conclusions in the literature (5–8) that implicate each of these pathways in the clearance of a fraction of α-synuclein.

Fig. 3.

Nedd4 accelerates the degradation of endogenous α-synuclein by the endosomal–lysosomal pathway. (A) In SH-SY5Y cells transfected with an empty vector, the degradation of endogenous α-synuclein decreased (and levels of α-synuclein increased) after treatment for 6 h with the proteasome inhibitor bortezomib (bort.; 10 μM), the lysosomal inhibitor chloroquine (CQ; 50 μM), or the autophagy inhibitor 3MA (10 mM). In contrast, when Nedd4 was overexpressed, the accelerated degradation of endogenous α-synuclein was blocked by chloroquine, but not by bortezomib or 3MA. (Upper) A typical experiment. (Lower) α-Synuclein quantification (n = 3). (B) α-Synuclein is ubiquitinated in cells expressing WT Nedd4, but not the inactive Nedd4 C-A mutant. (C) Control and Nedd4-expressing SH-SY5Y cells were cotransfected with either empty vector or RNAi for VPS24. At 72 h posttransfection, the level of endogenous α-synuclein was decreased in cells overexpressing Nedd4 in the presence of bortezomib (10 μM) or 3MA (10 mM) as above, and this effect was blocked only when the ESCRT three-component VPS24 was suppressed with RNAi. (D) HEK cells were transfected with siRNA against the ESCRT one-component tsg101 or scrambled (scr) siRNA for 24 h and then treated with cycloheximide (20 μg/mL) for the indicated times. When tsg101 was suppressed with siRNA, the rate of clearance of endogenous α-synuclein was reduced compared with control cells. (Lower) For quantification, the α-synuclein/actin band ratio was normalized to the corresponding ratio at T₀ on the same immunoblot (n = 3).

However, when α-synuclein degradation was accelerated by Nedd4 overexpression, this process was not reduced by blocking proteasomal function. In contrast, chloroquine treatment almost completely blocked the effect of Nedd4 on α-synuclein degradation (Fig. 3A). Thus, Nedd4 targets α-synuclein for hydrolysis by lysosomes and not by proteasomes. Blockage of autophagy with 3MA also failed to prevent the Nedd4-induced rapid degradation. To confirm that ubiquitination by Nedd4 targets α-synuclein to the lysosome, HEK cells expressing Nedd4, α-synuclein and HA-tagged ubiquitin and control HEK cells were treated with chloroquine to block lysosomal degradation. Ubiquitin conjugates were then immunoprecipitated from cell lysates with anti-HA antibodies and blotted with anti–α-synuclein antibodies. A high molecular weight smear, consistent with polyubiquitinated α-synuclein, was seen in cells overexpressing WT Nedd4, but not in control cells or cells expressing the active site mutant, Nedd4^C-A, which lacks ligase activity (Fig. 3B). Taken together, these findings indicate that, although autophagy and proteasomes contribute to basal turnover of α-synuclein, ubiquitination by Nedd4 accelerates α-synuclein degradation by a distinct lysosomal pathway.

Nedd4 Targets α-Synuclein for Degradation by the Endosomal-Sorting Complex Required for Transport Pathway.

It is now well established that conjugation of membrane-associated proteins, especially surface receptors, to K63-linked ubiquitin chains triggers their trafficking to endosomes and then to lysosomes by a series of steps catalyzed by the endosomal-sorting complex required for transport (ESCRT) 0–3 (20). To determine whether Nedd4 targets α-synuclein to lysosomes via the ESCRT pathway, we tested whether down-regulation of ESCRT components influences α-synuclein degradation. In neuroblastoma cells transfected with RNAi against vps24, a component of ESCRT3 (20), Nedd4 overexpression failed to enhance the rate of α-synuclein clearance. In contrast, the fraction of α-synuclein degraded by proteasomes (bortezomib-sensitive) and autophagy (3MA-sensitive) was unaffected (Fig. 3C). To further test whether trafficking of α-synuclein involves other ESCRT complexes and is similar in other cell types, we used RNAi to reduce tsg101 levels, a component of ESCRT1 (20), in HEK293 cells. After the addition of cycloheximide, the rate of clearance of α-synuclein was slower when tsg101 was down-regulated than in controls (Fig. 3D). These findings suggest that Nedd4 functions with endosomal vesicles in the breakdown of α-synuclein.

Nedd4 Ortholog Rsp5 Protects Against α-Synuclein in Yeast Models.

Saccharomyces cerevisiae-expressing α-synuclein has proven to be a valuable experimental system for identifying factors influencing α-synuclein toxicity that are relevant to mammalian cells (21–23). The Nedd4 ortholog in yeast, Rsp5p, is essential for viability, and like Nedd4 in mammals, it functions in the endosomal–vacuolar pathway, as well as in clearing damaged proteins (24, 25). We found that Rsp5p, like Nedd4, can bind α-synuclein in brain lysates (SI Appendix, Fig. S4) and efficiently ubiquitinates purified α-synuclein (Fig. 4A). Therefore, we asked whether Rsp5p, like Nedd4, promotes α-synuclein degradation and protects against α-synuclein–induced toxicity. To test whether Rsp5p increases the clearance of α-synuclein through ubiquitination, we used the temperature-sensitive rsp5-1 mutant (strain FW1808), which has a reduced ability to catalyze substrate ubiquitination (26) even at the permissive temperature. We measured the rate of α-synuclein degradation after a short induction in rsp5-1 cells transformed with either WT or an enzymatically inactive rsp5 mutant bearing a C→A mutation in its active site. α-Synuclein appeared stable for at least 4 h at the nonpermissive temperature, but restoration of a functional WT Rsp5p (not the inactive mutant form) increased the clearance of α-synuclein (Fig. 4 B and C).

Fig. 4.

In yeast, Rsp5p catalyzes α-synuclein ubiquitination and degradation and protects against inclusion formation and toxicity. (A) α-Synuclein is ubiquitinated similarly by purified Nedd4 and its yeast ortholog Rsp5p. (B) Restoration of a functional WT Rsp5p, but not of the inactive mutant form (Rsp5p C→A), increased the clearance of α-synuclein after short induction with galactose at the indicated time points. Equal amounts of total protein per lane was confirmed by Coomassie blue staining as shown. (C) Quantification of changes in α-synuclein levels (n = 3). (D) The accumulation of α-synuclein–GFP in inclusions was faster and greater in the Rsp5-1 mutants than in WT cells. (E) Expression of low-copy (Upper) and high-copy (Lower) α-synuclein plasmid in the Rsp5-1 strain is associated with greater toxicity than in WT. (F) Expression of Rsp5p in cells stably expressing α-synuclein reduces toxicity compared with yeast transfected with an empty vector.

In these strains, α-synuclein accumulation is often associated with the formation of intracellular inclusions, which contain transport vesicles and are believed to reflect a vesicular trafficking defect (21, 22). If Rsp5p targets α-synuclein to the vacuolar system in yeast, then reducing Rsp5p activity would be expected to enhance inclusion formation. To test this possibility, we transformed WT and rsp5-1 cells with GAL–α-synuclein–EGFP constructs and quantified the number of inclusions at different times after induction of α-synuclein expression. α-Synuclein–EGFP inclusions were three to four times more numerous and appeared sooner after induction in the rsp5-1 cells than in the WT cells (Fig. 4D). Thus, like Nedd4 in mammalian cells, Rsp5p promotes the clearance of α-synuclein through its ubiquitin-ligase activity. A major advantage of this yeast model is seen in studies assessing α-synuclein cytoxicity (22); thus, we used it to test whether Rsp5p affords protection against α-synuclein toxicity in these cells. Expression of α-synuclein in Rsp5-1 cells at 30 °C, where there is a partial loss of ubiquitin-ligase activity, caused a greater growth defect than in WT cells (Fig. 4E). Furthermore, using a strain that carries two copies of the GAL–α-synuclein plasmid incorporated in the genome and that has been used to screen for modifiers of toxicity (22), we found that expression of WT Rsp5p significantly reduced toxicity compared with control transformants (Fig. 4F). Thus, using two complementary approaches in different strains, we found that the ubiquitination activity of this Nedd4 ortholog decreases α-synuclein–induced toxicity.

Nedd4 Is Present in Pigmented Neurons of the Substantia Nigra and Locus Coeruleus and Is Strongly Expressed in Neurons with Lewy Bodies.

Because the neurodegenerative process shows a predilection for the dopaminergic and catecholaminergic pigmented neurons of the brainstem, we studied the distribution of Nedd4 in these neuronal populations in healthy control and disease states. In the pigmented neurons of the healthy substantia nigra, Nedd4 staining was seen primarily in neuronal processes (SI Appendix, Fig. S6). Accordingly, previous in situ hybridization studies in the mouse brain showed that Nedd4 and α-synuclein mRNA are highly expressed in this brain region (SI Appendix, Fig. S1). To confirm that these two proteins are expressed in the same neurons, we double-stained primary rat neurons in culture and found clear evidence of colocalization (SI Appendix, Fig. S6).

We then asked whether Nedd4 expression is altered in the brains of patients in whom α-synuclein accumulates in Lewy bodies. Using two different antibodies, we examined the pigmented neurons of the substantia nigra and locus coeruleus in a series of 13 well-characterized brains. In addition to classic PD, these included cases with Lewy bodies and mild nigral degeneration without overt clinical symptoms (termed incidental Lewy body disease), as well as diffuse Lewy body dementia (SI Appendix, Table S1). These tissues were compared with control brains lacking any pathological evidence of Lewy body degeneration (n = 4). In contrast to neurons in control brains, in all cases with Lewy body pathology, Nedd4 was strongly expressed in a subpopulation of pigmented neurons in both of these brain areas (Fig. 5 A and B). In control experiments, in which the primary antibody was omitted, no staining was seen (Fig. 5C). Nedd4 distribution ranged from diffuse strong staining in dysmorphic neurons without obvious compact aggregates (Fig. 5 D–F) to a striking ring-shaped localization around Lewy bodies (Fig. 5 G–I). To further document this association, we quantified this pathological staining pattern in the locus coeruleus. It was detected in 43% ± 6.7% (range, 15–80%) of neurons in cases with Lewy body pathology and was not evident in controls. Occasional Nedd4 staining was also seen in the adjacent reactive glia (Fig. 5D, arrowhead) and dystrophic neurites (Fig. 5H, arrows).

Fig. 5.

Nedd4 is strongly expressed in pigmented neurons with Lewy bodies. The locus coeruleus and the substantia nigra were assessed in a series of 13 cases with Lewy body pathology and 4 healthy controls (SI Appendix, Table S1). (A) In cases with Lewy body pathology, Nedd4 is strongly expressed in a subpopulation of pigmented neurons. (B) In contrast, in healthy control brains, Nedd4 is not detected in the cell soma of pigmented neurons. (C) No staining is detected in sections without primary antibody. (D–I) High magnification in the substantia nigra and locus coeruleus show that Nedd4 is either diffusely expressed in neurons without compact aggregates (D–F) or localized in a ring-like pattern around Lewy bodies (G–I). Note the occasional reactive glia (arrowhead in D); adjacent, presumably healthy neurons without staining (F and G); and Nedd4-positive dystrophic processes (arrows in H). (J–L) High magnification of a pigmented neuron in the substantia nigra from Parkinson disease brain (J) stained positive for Lewy bodies (K, green) and Nedd4 (L, red). (M) Superimposed images showing Nedd4 in Lewy body-positive neurons. (Scale bar: 40 μm.)

Discussion

Together, our findings indicate that in human cells, ubiquitination by Nedd4 is an important factor in promoting the destruction of α-synuclein by the endosomal–lysosomal pathway. This pathway has not been previously implicated in α-synuclein turnover, even though this protein is membrane-associated and is localized primarily to synaptic terminals, where its level influences vesicular recycling and increased levels can impair neurotransmitter release (3). Involvement of the endosomal–lysosomal pathway in the clearance of α-synuclein by Nedd4 is supported by the requirement for ESCRT complexes, by its sensitivity to lysosomotropic inhibitors (but not to inhibitors of proteasomes or autophagy), and by the linkage to α-synuclein of K63 ubiquitin chains. Thus, it is possible that the endosomal–lysosomal pathway specifically catalyzes the degradation of a membrane-associated pool of α-synuclein, whereas proteasomes and autophagy, which also play a role in α-synuclein turnover (Fig. 3A), degrade the soluble and aggregated forms, respectively. Accordingly, α-synuclein can be detected in lysosomes of normal brain, and blockage of autophagy only partially prevented this localization (28).

Other observations also support an important role for the endosomal-lysosomal system. This pathway has been highly conserved from yeast to human cells, and Nedd4 and Rsp5p clearly show similar substrate specificities and enzymatic activities despite the evolutionary distance between them. Of note, mutations in components of the endosomal pathway in yeast increase susceptibility to α-synuclein toxicity (29), as was found in the present study with mutations affecting the ubiquitination activity of Rsp5p. Similarly, in C. elegans, α-synuclein expression causes toxicity that is enhanced by down-regulation of components of the endosomal pathway (30). Importantly, recent genome-wide association studies have led to the identification of an SNP in Nedd4 as a risk factor for PD (31), and have uncovered genes that encode components of the endocytic–lysosomal pathway (32). It is also noteworthy that mutations in glucocerebrosidase, the lysosomal enzyme defective in Gaucher's disease, carry a fivefold increased risk of PD with Lewy bodies (33).

Biochemical analysis of ubiquitinated components in Lewy bodies is complicated by the rapid deubiquitination of proteins in postmortem samples. Furthermore, multiple deubiquitinating enzymes function in the endosomal targeting of membrane proteins to lysosomes. Nevertheless, it is noteworthy that in the present study, purified Nedd4 formed ubiquitin chains on lysine 21, which has been shown to be a major site of α-synuclein ubiquitination in Lewy bodies (34).

Although further studies of animal models are essential to clarify the precise role of the Nedd4–endosomal pathway in neuroprotection, the dramatic finding that Nedd4 is expressed especially strongly in pigmented neurons with Lewy body pathology in the locus coeruleus and substantia nigra suggests an important role for this enzyme in human disease. Previous studies also indicated that expression of Nedd4 in neurons is tightly regulated. Its expression is induced in the brain during neurogenesis (27) and after traumatic brain injury (35). Although it is possible that this build-up of Nedd4 in neurons with Lewy bodies is a consequence of neuronal damage, it seems more likely that up-regulation of Nedd4 represents a protective response that helps reduce the accumulation of this potentially toxic protein. Accordingly, our studies indicate that Rsp5p affords protection against α-synuclein toxicity in yeast, and that endogenous Nedd4 reduces α-synuclein accumulation in human cells. Of note, in transgenic mice and Drosophila models, expression of truncated α-synuclein 1–120, which cannot be ubiquitinated by Nedd4, is associated with accelerated pathology and ubiquitin-negative inclusions (36, 37). It thus seems likely that in Lewy body disease, activation of the Nedd4-endosomal pathway is an important cellular defense mechanism that enhances α-synuclein clearance. Consequently, even though Nedd4 has multiple physiological substrates, pharmacologic activation or induction of Nedd4 in neurons may represent a possible therapeutic approach to retard the progression of PD and other α-synucleinopathies.

Methods

Ubiquitination Assays.

WT human α-synuclein protein (untagged full-length and fragments) was purified as described previously (SI Appendix). Purified α-synuclein was added to the ubiquitination mixture containing 6His-E1 (50 nM), UbcH5 or UbcH7 (750 nM), 6His-E3 (500 nM), and ubiquitin (59 mM) in a buffer composed of 20 mM Tris-HCl (pH 7.6), 20 mM KCl, 5 mM MgCl₂, 2 mM ATP, and 1 mM DTT. Ubiquitination reactions were performed at 30 °C for the indicated times.

LC-MS Analysis and Database Search.

After in-gel trypsinization of ubiquitinated α-synuclein, the digested peptides were loaded onto a fused silica microcapillary column, packed with C18 resin (5 mm, 300 Å; Alltech), and separated by HPLC (Eksigent). The column was connected directly to an LTQ XL ion-trap mass spectrometer (Thermo Scientific) as described in SI Appendix. For the database search, all MS/MS spectra recorded were searched using the SEQUEST algorithm (Thermo Finnigan) and TurboSEQUEST (Thermo Electron) and Scaffold version 3_00_02 (Proteome Software). To quantify the polyubiquitinated chain, AQUA analysis was performed for three ubiquitinated peptides on Ub (SI Appendix, Table S2).

Assays in Yeast.

For viability, cultures were grown until they reached midlog phase, then normalized for OD₆₀₀, serially diluted, and spotted onto solid media containing either 2% glucose (α-synuclein off) or 2% galactose (α-synuclein on), after which they were grown at 30 °C for 2–3 d. For degradation, equal amount of cells were induced at 30 °C with 2% (wt/vol) galactose, washed in 2% (wt/vol) glucose twice (to switch off α-synuclein expression), and incubated in medium containing 2% (wt/vol) glucose for the indicated time points at 37 °C. Equal amounts of cells were collected, centrifuged, and placed on dry ice until further use.

Staining of Human Sections.

Human sections through the substantia nigra and locus coeruleus from Lewy body disease and control brains (SI Appendix, Table S1) were kindly provided by the Thomas Willis Brain Bank, Oxford University. Sections were dewaxed, autoclaved, and incubated with rabbit anti-Nedd4 antibodies (Abcam) overnight at 4 °C. Primary antibody detection was performed using biotin-free indirect labeling. For immunofluorescence, sections were permeabilized in 0.5% Triton ×100, blocked in serum, and incubated with anti-Nedd4 polyclonal (Abcam) and syn-1 monoclonal (BD Biosciences) antibodies overnight at 4 °C. Sections were incubated with appropriate Alexa Fluor secondary antibodies (Molecular Probes).

Cell Culture.

SH-SY5Y (American Type Culture Collection) human dopaminergic neuroblastoma or HEK cell lines were used. Cells were maintained at 37 °C and 5% CO₂ in DMEM/F12 (DMEM/Ham's F-12 medium, 50/50 mix) for SH-SY5Y cells or in DMEM for HEK cells with l-glutamine, supplemented with 10% (vol/vol) FBS (Sigma-Aldrich) and 1% (vol/vol) penicillin/streptomycin (Cellgro; Mediatech). Cells were grown to 60–80% confluency and transfected using ExGen500 (Fermentas) and 4 μg of plasmid DNA. Between 24 and 48 h after transfection, cells were treated with 1 μM Velcade (a gift from Millenium), 10 mM 3MA (Sigma-Aldrich), or 50 μM chloroquine (Sigma-Aldrich). To inhibit protein synthesis, cells were treated with 20 μg/mL cycloheximide (Sigma-Aldrich). The plasmid used to overexpress Nedd4 consisted of full-length rat Nedd4 and C-A mutant T7 epitope-tagged placed at the N terminus (a kind gift from Dr, Daniela Rotin, University of Toronto).