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.

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 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).

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.

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.)