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 ...

α-Synuclein accumulation is a pathological hallmark of Parkinson's disease (PD). Ubiquitinated α-synuclein is targeted to proteasomal or lysosomal degradation. Here, we identify SUMOylation as a major mechanism that counteracts ubiquitination by different E3 ubiquitin ligases and regulates α-synuclein degradation. We report that PIAS2 promotes SUMOylation of α-synuclein, leading to a decrease in α-synuclein ubiquitination by SIAH and Nedd4 ubiquitin ligases, and causing its accumulation and aggregation into inclusions. This was associated with an increase in α-synuclein release from the cells. A SUMO E1 inhibitor, ginkgolic acid, decreases α-synuclein levels by relieving the inhibition exerted on α-synuclein proteasomal degradation. α-Synuclein disease mutants are more SUMOylated compared with the wild-type protein, and this is associated with increased aggregation and inclusion formation. We detected a marked increase in PIAS2 expression along with SUMOylated α-synuclein in PD brains, providing a causal mechanism underlying the up-regulation of α-synuclein SUMOylation in the disease. We also found a significant proportion of Lewy bodies in nigral neurons containing SUMO1 and PIAS2. Our observations suggest that SUMOylation of α-synuclein by PIAS2 promotes α-synuclein aggregation by two mutually reinforcing mechanisms. First, it has a direct proaggregatory effect on α-synuclein. Second, SUMOylation facilitates α-synuclein aggregation by blocking its ubiquitin-dependent degradation pathways and promoting its accumulation. Therefore, inhibitors of α-synuclein SUMOylation provide a strategy to reduce α-synuclein levels and possibly aggregation in PD.

α-Synuclein misfolding and aggregation is a hallmark in Parkinson's disease and in several other neurodegenerative diseases known as synucleinopathies. The toxic properties of α-synuclein are conserved from yeast to man, but the precise underpinnings of the cellular pathologies associated are still elusive, complicating the development of effective therapeutic strategies. Combining molecular genetics with target-based approaches, we established that glycation, an unavoidable age-associated post-translational modification, enhanced α-synuclein toxicity in vitro and in vivo, in Drosophila and in mice. Glycation affected primarily the N-terminal region of α-synuclein, reducing membrane binding, impaired the clearance of α-synuclein, and promoted the accumulation of toxic oligomers that impaired neuronal synaptic transmission. Strikingly, using glycation inhibitors, we demonstrated that normal clearance of α-synuclein was re-established, aggregation was reduced, and motor phenotypes in Drosophila were alleviated. Altogether, our study demonstrates glycation constitutes a novel drug target that can be explored in synucleinopathies as well as in other neurodegenerative conditions.

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...

The mechanism of RNA degradation in Escherichia coli involves endonucleolytic cleavage, polyadenylation of the cleavage product by poly(A) polymerase, and exonucleolytic degradation by the exoribonucleases, polynucleotide phosphorylase (PNPase) and RNase II. The poly(A) tails are homogenous, containing only adenosines in most of the growth conditions. In the chloroplast, however, the same enzyme, PNPase, polyadenylates and degrades the RNA molecule; there is no equivalent for the E. coli poly(A) polymerase enzyme. Because cyanobacteria is a prokaryote believed to be related to the evolutionary ancestor of the chloroplast, we asked whether the molecular mechanism of RNA polyadenylation in the Synechocystis PCC6803 cyanobacteria is similar to that in E. coli or the chloroplast. We found that RNA polyadenylation in Synechocystis is similar to that in the chloroplast but different from E. coli. No poly(A) polymerase enzyme exists, and polyadenylation is performed by PNPase, resulting in heterogeneous poly(A)-rich tails. These heterogeneous tails were found in the amino acid coding region, the 5 and 3 untranslated regions of mRNAs, as well as in rRNA and the single intron located at the tRNA fmet . Furthermore, unlike E. coli, the inactivation of PNPase or RNase II genes caused lethality. Together, our results show that the RNA polyadenylation and degradation mechanisms in cyanobacteria and chloroplast are very similar to each other but different from E. coli.The molecular mechanism of RNA degradation in prokaryotes and organelles includes a series of sequential steps. The degradation starts with the initial endonucleolytic cleavage carried out primarily by the endoribonuclease E (RNase E). The cleavage products are then polyadenylated at their 3Ј ends by the poly(A) polymerase (PAP) 1 enzyme in Escherichia coli and the polynucleotide phosphorylase (PNPase) in the chloroplast. The polyadenylated molecules are then rapidly degraded exonucleolytically by PNPase and ribonuclease II (RNase II) (1-3). Finally, the remaining short oligoribonucleotides are degraded by the oligoribonuclease enzyme (4). Although the inhibition of polyadenylation in the chloroplast inhibited exonucleolytic degradation, implying that this is the only mechanism in the RNA degradation process, two RNA degradation mechanisms, a polyadenylation-dependent and a polyadenylation-independent one, were suggested to take place in E. coli (5, 6). Polyadenylation in E. coli is carried out primarily by PAP (7). Also in this bacterium, the RNase E enzyme, part of the PNPase population, an RNA helicase, some RNA molecules, and the glycolytic enzyme enolase are associated in a high molecular weight complex called a degradosome (8).In the chloroplast, the photosynthetic organelle of the plant cell is believed to have an evolutionary prokaryotic origin; many characteristics of the gene expression system resemble those of bacteria. When the RNA degradation mechanism was analyzed in the chloroplast, it was found to be very similar to that of E. coli (1-3). However...

PTEN-induced putative kinase 1 (PINK1) and parkin are mutated in familial forms of Parkinson's disease and are important in promoting the mitophagy of damaged mitochondria. In this study, we showed that synphilin-1 interacted with PINK1 and was recruited to the mitochondria. Once in the mitochondria, it promoted PINK1-dependent mitophagy, as revealed by Atg5 knockdown experiments and the recruitment of LC3 and Lamp1 to the mitochondria. PINK1-synphilin-1 mitophagy did not depend on PINK1-mediated phosphorylation of synphilin-1 and occurred in the absence of parkin. Synphilin-1 itself caused depolarization of the mitochondria and increased the amount of uncleaved PINK1 at the organelle. Furthermore, synphilin-1 recruited seven in absentia homolog (SIAH)-1 to the mitochondria where it promoted mitochondrial protein ubiquitination and subsequent mitophagy. Mitophagy via this pathway was impaired by synphilin-1 knockdown or by the use of a synphilin-1 mutant that is unable to recruit SIAH-1 to the mitochondria. Likewise, knockdown of SIAH-1 or the use of a catalytically inactive SIAH-1 mutant abrogated mitophagy. PINK1 disease mutants failed to recruit synphilin-1 and did not activate mitophagy, indicating that PINK1-synphilin-1-SIAH-1 represents a new parkin-independent mitophagy pathway. Drugs that activate this pathway will provide a novel strategy to promote the clearance of damaged mitochondria in Parkinson's disease.

Mutations in Parkin are responsible for a large percentage of autosomal recessive juvenile parkinsonism cases. Parkin displays ubiquitin-ligase activity and protects against cell death promoted by several insults. Therefore, regulation of Parkin activities is important for understanding the dopaminergic cell death observed in Parkinson disease. We now report that cyclin-dependent kinase 5 (Cdk5) phosphorylates Parkin both in vitro and in vivo. We found that highly specific Cdk5 inhibitors and a dominant negative Cdk5 construct inhibited Parkin phosphorylation, suggesting that a significant portion of Parkin is phosphorylated by Cdk5. Parkin interacts with Cdk5 as observed by co-immunoprecipitation experiments of transfected cells and rat brains. Phosphorylation by Cdk5 decreased the auto-ubiquitylation of Parkin both in vitro and in vivo. We identified Ser-131 located at the linker region of Parkin as the major Cdk5 phosphorylation site. The Cdk5 phosphorylationdeficient S131A Parkin mutant displayed a higher auto-ubiquitylation level and increased ubiquitylation activity toward its substrates synphilin-1 and p38. Additionally, the S131A Parkin mutant more significantly accumulated into inclusions in human dopaminergic cells when compared with the wild-type Parkin. Furthermore, S131A Parkin mutant increased the formation of synphilin-1/␣-synuclein inclusions, suggesting that the levels of Parkin phosphorylation and ubiquitylation may modulate the formation of inclusion bodies relevant to the disease. The data indicate that Cdk5 is a new regulator of the Parkin ubiquitin-ligase activity and modulates its ability to accumulate into and modify inclusions. Phosphorylation by Cdk5 may contribute to the accumulation of toxic Parkin substrates and decrease the ability of dopaminergic cells to cope with toxic insults in Parkinson disease. Parkinson disease (PD)2 is one of the most common neurodegenerative diseases. It is characterized by a progressive degeneration of dopaminergic neurons in the substantia nigra and the presence of cytoplasmic inclusions called Lewy bodies in surviving neurons (1).The large majority of PD cases are sporadic, but six genes have been found to be mutated in familial forms of PD (1-3). Mutations in Parkin are the most frequent cause of familial PD and are responsible for a large percent of autosomal recessive juvenile parkinsonism (AR-JP) (4). This juvenile form of the disease is characterized by the death of dopaminergic neurons in the substantia nigra with the absence of Lewy bodies in surviving neurons, raising the possibility that Parkin may be essential for the formation of Lewy bodies (1, 5). Parkin may also play a role in sporadic PD, as several studies demonstrated that Parkin immunoreactivity is present in Lewy bodies (6 -8).Parkin has a ubiquitin-like domain at the N terminus connected to the C-terminal RING finger domains by a linker region (1). The presence of the RING finger domains confers to Parkin the ability to work as an E3 ubiquitin-ligase enzyme (9, 10). On the other hand...