␣-Synuclein plays a major role in Parkinson disease. Unraveling the mechanisms of ␣-synuclein aggregation is essential to understand the formation of Lewy bodies and their involvement in dopaminergic cell death. ␣-Synuclein is ubiquitylated in Lewy bodies, but the role of ␣-synuclein ubiquitylation has been mysterious. We now report that the ubiquitin-protein isopeptide ligase seven in absentia homolog (SIAH) directly interacts with and monoubiquitylates ␣-synuclein and promotes its aggregation in vitro and in vivo, which is toxic to cells. Mass spectrometry analysis demonstrates that SIAH monoubiquitylates ␣-synuclein at lysines 12, 21, and 23, which were previously shown to be ubiquitylated in Lewy bodies. SIAH ubiquitylates lysines 10, 34, 43, and 96 as well. Suppression of SIAH expression by short hairpin RNA to SIAH-1 and SIAH-2 abolished ␣-synuclein monoubiquitylation in dopaminergic cells, indicating that endogenous SIAH ubiquitylates ␣-synuclein. Moreover, SIAH co-immunoprecipitated with ␣-synuclein from brain extracts. Inhibition of proteasomal, lysosomal, and autophagic pathways, as well as overexpression of a ubiquitin mutant less prone to deubiquitylation, G76A, increased monoubiquitylation of ␣-synuclein by SIAH. Monoubiquitylation increased the aggregation of ␣-synuclein in vitro. At the electron microscopy level, monoubiquitylated ␣-synuclein promoted the formation of massive amounts of amorphous aggregates. Monoubiquitylation also increased ␣-synuclein aggregation in vivo as observed by increased formation of ␣-synuclein inclusion bodies within dopaminergic cells. These inclusions are toxic to cells, and their formation was prevented when endogenous SIAH expression was suppressed. Our data suggest that monoubiquitylation represents a possible trigger event for ␣-synuclein aggregation and Lewy body formation. Parkinson disease (PD)3 is characterized by progressive degeneration of dopaminergic neurons in the substantia nigra and the presence of cytoplasmic inclusions called Lewy bodies in surviving neurons (1).The majority of PD cases are sporadic, but mutations in different genes have been found to be responsible for familial PD (1, 2). ␣-Synuclein is believed to play a crucial role in the disease because it is mutated in some familial forms of the disease, and it is a major component of Lewy bodies in sporadic PD (3, 4). Three missense mutations in the ␣-synuclein gene, leading to A53T, A30P, and E46K substitutions at the protein level, have been described so far (5-7). Duplication and triplication of the ␣-synuclein gene were also shown to cause disease, suggesting that increase in the levels of ␣-synuclein can be pathogenic (8 -10). In agreement, mouse models overexpressing ␣-synuclein revealed a correlation between ␣-synuclein accumulation and neuronal dysfunction (11-13). Drosophila models also recapitulate some features of the disease, such as aggregation of ␣-synuclein and death of dopaminergic neurons (14, 15). Nonetheless, the absence of classical Lewy bodies in some live models suggests ...

α-Synuclein is central to the pathogenesis of Parkinson disease (PD). Mutations as well as accumulation of α-synuclein promote the death of dopaminergic neurons and the formation of Lewy bodies. α-Synuclein is monoubiquitinated by SIAH, but the regulation and roles of monoubiquitination in α-synuclein biology are poorly understood. We now report that the deubiquitinase USP9X interacts in vivo with and deubiquitinates α-synuclein. USP9X levels are significantly lower in cytosolic fractions of PD substantia nigra and Diffuse Lewy Body disease (DLBD) cortices compared to controls. This was associated to lower deubiquitinase activity toward monoubiquitinated α-synuclein in DLBD cortical extracts. A fraction of USP9X seems to be aggregated in PD and DLBD, as USP9X immunoreactivity is detected in Lewy bodies. Knockdown of USP9X expression promotes accumulation of monoubiquitinated α-synuclein species and enhances the formation of toxic α-synuclein inclusions upon proteolytic inhibition. On the other hand, by manipulating USP9X expression levels in the absence of proteolytic impairment, we demonstrate that monoubiquitination controls the partition of α-synuclein between different protein degradation systems. Deubiquitinated α-synuclein is mostly degraded by autophagy, while monoubiquitinated α-synuclein is preferentially degraded by the proteasome. Moreover, monoubiquitination promotes the degradation of α-synuclein, whereas deubiquitination leads to its accumulation, suggesting that the degradation of deubiquitinated α-synuclein by the autophagy pathway is less efficient than the proteasomal one. Lower levels of cytosolic USP9X and deubiquitinase activity in α-synucleinopathies may contribute to the accumulation and aggregation of monoubiquitinated α-synuclein in Lewy bodies. Our data indicate that monoubiquitination is a key determinant of α-synuclein fate. P arkinson disease (PD) is mainly characterized by the death of dopaminergic neurons in the substantia nigra and presence of inclusions called Lewy bodies. Even though Lewy bodies are composed predominantly of α-synuclein, the mechanisms by which Lewy bodies are formed and their role in the death of dopaminergic neurons remain elusive (1, 2).α-Synuclein purified from Lewy bodies is monoubiquitinated (3-5). Although some ubiquitin-ligases can polyubiquitinate α-synuclein (6-8), the E3 ubiquitin-ligase responsible for α-synuclein monoubiquitination has remained obscure. We and others have recently shown that α-synuclein is monoubiquitinated by the E3 ubiquitin-ligase SIAH both in vitro and in vivo (9-11). SIAH is present in Lewy bodies (9) and monoubiquitinates α-synuclein at the same lysine residues that are monoubiquitinated in α-synuclein immunopurified from Lewy bodies (5, 10). Most importantly, we found that in the presence of proteolytic inhibitors, monoubiquitination of α-synuclein promoted by SIAH leads to a marked increase in the aggregation of α-synuclein and formation of inclusions, which are toxic to cells (10-12). Therefore, our data indicate that monou...

␣-Synucleinopathies are a group of neurological disorders characterized by the presence of intracellular inclusion bodies containing ␣-synuclein. We previously demonstrated that synphilin-1 interacts with ␣-synuclein, implying a role in Parkinson's disease. We now report the identification and characterization of synphilin-1A, an isoform of synphilin-1, which has enhanced aggregatory properties and causes neurotoxicity. The two transcripts encoding synphilin-1A and synphilin-1 originate from the SNCAIP gene but differ in both their exon organization and initial reading frames used for translation. Synphilin-1A binds to ␣-synuclein and induces the formation of intracellular aggregates in human embryonic kidney 293 cells, primary neuronal cultures, and human dopaminergic cells. Overexpression of synphilin-1A in neurons results in striking cellular toxicity that is attenuated by the formation of synphilin-1A inclusions, which recruit ␣-synuclein. Synphilin-1A is present in Lewy bodies of patients with Parkinson's disease and Diffuse Lewy Body disease, and is observed in detergent-insoluble fractions of brain protein samples obtained from Diffuse Lewy Body disease patients. These findings suggest that synphilin-1A may contribute to neuronal degeneration in ␣-synucleinopathies and also provide important insights into the role of inclusion bodies in neurodegenerative disorders.␣-synuclein ͉ cell death ͉ Lewy body ͉ Parkinson's disease