Parkinson's disease (PD) is a neurodegenerative disease characterized by Lewy body formation and death of dopaminergic neurons. Mutations in ␣-synuclein and parkin cause familial forms of PD. Synphilin-1 was shown to interact with ␣-synuclein and to promote the formation of cytosolic inclusions. We now report that synphilin-1 interacts with the E3 ubiquitin-ligases SIAH-1 and SIAH-2. SIAH proteins ubiquitylate synphilin-1 both in vitro and in vivo, promoting its degradation by the ubiquitin-proteasome system. Inability of the proteasome to degrade synphilin-1͞SIAH complex leads to a robust formation of ubiquitylated cytosolic inclusions. Ubiquitylation is required for inclusion formation, because a catalytically inactive mutant of SIAH-1, which still binds to synphilin-1, fails to promote inclusions. Like synphilin-1, ␣-synuclein associates with SIAH in intact cells, but the interaction with SIAH-2 was much stronger that with SIAH-1. In vitro experiments show that SIAH-2 monoubiquitylates ␣-synuclein. Further evidence that SIAH proteins may play a role in inclusion formation comes from the demonstration of SIAH immunoreactivity in Lewy bodies of PD patients.␣-synuclein-interacting protein ͉ ubiquitin ͉ inclusion bodies

␣-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 ...