Human Molecular Genetics

Human Molecular Genetics Advance Access originally published online on December 7, 2007
Human Molecular Genetics 2008 17(6):906-917; doi:10.1093/hmg/ddm363

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Ubiquitination of -synuclein by Siah-1 promotes -synuclein aggregation and apoptotic cell death

James T. Lee¹, Tiffany C. Wheeler², Lian Li^1,2 and Lih-Shen Chin^1,2,*

¹ Department of Pharmacology, Center for Neurodegenerative Disease, Emory University School of Medicine, Atlanta, GA 30322-3090, USA ² Department of Pharmacology, School of Medicine, University of North Carolina, Chapel Hill, NC 27599, USA

^* To whom correspondence should be addressed. Tel: +1 4047270361; Fax: +1 4047270365; Email: chinl{at}pharm.emory.edu

Received August 23, 2007; Revised October 31, 2007; Accepted December 5, 2007

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS FUNDING REFERENCES

 
Point mutations and gene multiplication of -synuclein cause autosomal dominant familial Parkinson's disease (PD). Moreover, -synuclein- and ubiquitin-positive inclusion bodies are the pathological hallmarks of PD and several other neurodegenerative diseases, such as dementia with Lewy bodies and multiple system atrophy. Despite the presence of ubiquitinated -synuclein species in Lewy bodies, the regulation of -synuclein ubiquitination and its role in Lewy body formation and neurodegeneration remain poorly understood. Here, we report that -synuclein interacts and colocalizes with mammalian seven in absentia homologue-1 (Siah-1), a RING-type E3 ubiquitin-protein ligase. Siah-1 binds the brain-enriched E2 ubiquitin-conjugating enzyme UbcH8 and facilitates mono- and di-ubiquitination of -synuclein in vivo. The ubiquitination of -synuclein by Siah-1 is disrupted by the PD-linked A30P mutation but not by A53T mutation. We find that Siah-1-mediated ubiquitination does not target -synuclein for degradation by the proteasome, but rather, it promotes -synuclein aggregation and enhances -synuclein toxicity. Our findings suggest that Siah-1-mediated -synuclein ubiquitination may play a critical role in Lewy body formation and PD pathogenesis.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS FUNDING REFERENCES

 
Parkinson's disease (PD) is the most common neurodegenerative movement disorder, characterized by the loss of dopaminergic neurons in the substantia nigra and the presence of intracellular inclusions known as Lewy bodies (1,2). Although the cause of neurodegeneration in PD remains unclear, genetic studies have identified several genes responsible for rare familial forms of PD (3). The findings that single point mutations (A53T and A30P) in the -synuclein gene causes autosomal dominant form of familial PD (4,5) provide first evidence directly linking -synuclein to PD. The recent identification of -synuclein gene duplication and triplication as a cause for familial PD (6–8) further indicates an association between the increased expression of wild-type -synuclein and PD pathogenesis. Moreover, -synuclein is a major component of Lewy bodies in brains of patients with sporadic PD (9,10) and -synuclein-containing inclusion bodies are pathological hallmarks of several other neurodegenerative diseases (collectively referred to as -synucleinopathies), including dementia with Lewy bodies and multiple system atrophy (11). Elucidation of the molecular mechanisms that control -synuclein aggregation and inclusion formation is thus crucial for understanding the pathogenesis of PD and related neurodegenerative diseases.

Lewy bodies and other -synuclein-containing inclusions in -synucleinopathies are immunoreactive to anti-ubiquitin antibodies (12,13). Recent evidence indicates that -synuclein is ubiquitinated in Lewy bodies (14,15). Interestingly, the ubiquitinated -synuclein species found in Lewy bodies are mono- and di-ubiquitinated forms rather than the polyubiquitinated form associated with proteasomal degradation (14–17). The functional consequence of -synuclein mono- and di-ubiquitination and its role in Lewy body formation and neurodegeneration remain undefined.

Dysfunction of the ubiquitin-proteasome system has been strongly implicated in PD pathogenesis (18–20). Mutations in the E3 ubiquitin-protein ligase parkin cause recessively transmitted early-onset form of PD (21–23). Parkin has been shown to polyubiquitinate O-glycosylated -synuclein (Sp22), a rare form of -synuclein (24). However, parkin does not ubiquitinate non-glycosylated -synuclein (25), which is the predominant -synuclein species in brain. The existence of ubiquitinated non-glycosylated -synuclein species in Lewy bodies (14) suggests that another E3 ligase is responsible for ubiquitination of non-glycosylated -synuclein.

We have previously characterized mammalian homologues of Drosophila Seven in Absentia (Sina), Siah-1 and Siah-2, as a family of RING-type E3 ligases that regulate ubiquitination and degradation of presynaptic protein synaptophysin (26). Siah-1 and Siah-2 are encoded by two distinct genes, and their protein sequences exhibit 77% amino acid identity (27). Both Siah proteins are expressed in the brain as well as many other tissues, although Siah-1 appears to be more abundantly expressed than Siah-2 (27–30). A recent study shows that Siah-1, but not Siah-2, is present in Lewy bodies found in substantia nigra of PD patients (31). Despite similar domain structure and high degree of sequence homology, Siah-1 and Siah-2 do not always have the same substrates (32), suggesting that some biochemical functions of Siah proteins are unique to each family members. In support of this view, targeted disruption of Siah-1 gene expression in mice results in postnatal growth retardation and premature death (33), whereas Siah-2 knockout mice are largely phenotypically normal (34). Recently, it was reported that -synuclein is a substrate of Siah-2 (31). However, whether -synuclein can be ubiquitinated by Siah-1 remains unclear (31,35) and the cellular role of -synuclein ubiquitination is unknown.

In this study, we investigated the role of Siah-1 in ubiquitinating -synuclein and the functional consequences of Siah-1-mediated -synuclein ubiquitination. Our results indicate that Siah-1 facilitates mono- and di-ubiquitination of -synuclein, and the Siah-1-mediated ubiquitination promotes -synuclein aggregation and apoptotic cell death. These findings have important implications for understanding Lewy body formation and neurodegeneration in PD.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS FUNDING REFERENCES

 
Identification of -synuclein as a Siah-1-interacting protein
We have previously shown that Siah-1 E3 ligase binds and ubiquitinates synaptic vesicle protein synaptophysin (26). To identify additional synaptic substrates of Siah-1, we used a GST-Siah-1 column to pull down brain proteins and screen bound proteins using antibodies against various synaptic vesicle-associated proteins, such as synaptobrevin, synaptotagmin and synuclein. We found that purified GST-Siah-1, but not the GST control, was able to affinity-purify endogenous -synuclein from rat brain homogenates (Fig. 1A), suggesting an interaction between Siah-1 and -synuclein. To further confirm this interaction, we performed co-immunoprecipitation experiments using HeLa cells co-transfected with pMyc--synuclein and pCHA-Siah-1 or pCHA vector. Immunoprecipitation of transfected cell lysates with anti-HA antibody revealed that Myc--synuclein was specifically co-immunoprecipitated with HA-Siah-1, but was not co-immunoprecipitated in vector-transfected cells (Fig. 1B). In addition, we performed co-immunoprecipitation analysis with anti--synuclein antibody to examine the interaction between endogenous -synuclein and Siah-1 in PC12 cells. We found that endogenous Siah-1 co-immunoprecipitated with -synuclein (Fig. 1C), indicating that Siah-1 associates with -synuclein in vivo.

View larger version (27K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 1. Siah-1 interacts with -synuclein in vitro and in vivo. (A) In vitro binding assays were performed by incubation of immobilized GST or GST-Siah-1 fusion proteins (lower panel, Ponceau S staining) with rat brain homogenate (Input). Bound -synuclein was detected by immunoblotting for -synuclein (upper panel). (B) -Synuclein co-immunoprecipitates with Siah-1 in transfected HeLa cells. HeLa cells were co-transfected with pMyc--synuclein and pCHA vector or pCHA-Siah-1, and the cell lysates were immunoprecipitated with anti-HA antibody followed by immunoblotting with anti-Myc and anti-HA antibodies. The asterisk indicates a degradation product of HA-Siah-1. (C) Co-immunoprecipitation of endogenous Siah-1 with -synuclein in PC12 cells. PC12 cell lysates were immunoprecipitated with anti--synuclein antibody or control mouse IgG followed by immunoblotting for -synuclein and Siah-1.

 
A subpopulation of endogenous Siah-1 colocalizes with -synuclein
To provide further evidence for an in vivo association between -synuclein and Siah-1, we performed double immuno fluorescence labeling experiments to examine whether endogenous -synuclein colocalizes with Siah-1. In agreement with our previous finding that a portion of Siah-1 is membrane-associated (26), we observed a punctate staining pattern of endogenous Siah-1 in NGF-differentiated PC12 cells (Fig. 2A) as well as in primary mouse cortical neurons (Fig. 2B and C). The intracellular distribution of Siah-1 showed a significant overlap with that of -synuclein in the cell bodies and neuritic processes (Fig. 2), indicating that at least a subpopulation of endogenous Siah-1 colocalizes with -synuclein in PC12 cells as well as in cortical neurons.

View larger version (62K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 2. Analysis of -synuclein and Siah-1 colocalization by immunofluorescence confocal microscopy. (A and B) NGF-differentiated PC12 cells (A) and primary cortical neurons (B) were stained for Siah-1 (red) and -synuclein (green). Insets: enlarged views of boxed area in the cell body showing substantial colocalization between endogenous -synuclein and Siah-1. (C) An enlarged view (3x) of the boxed region in (B) highlights colocalization between Siah-1 and -synuclein in neurites. Arrowheads indicate regions of colocalization between Siah-1 and -synuclein. Scale bar=10 µm in (A) and 20 µm in (B).

 
Siah-1 facilitates mono- and di-ubiquitination of -synuclein
To identify cognate E2 ubiquitin-conjugating enzyme(s) for Siah-1 E3 ligase, we expressed HA-tagged E2 enzymes UbcH5, UbcH7 and UbcH8 by transient transfection into HeLa cells along with Myc-tagged Siah-1. The interaction of these E2 enzymes with Siah-1 was examined by immunoprecipitation with an anti-Myc antibody followed by immunoblotting with an anti-HA antibody (Fig. 3A). The results reveal that Siah-1 specifically interacts with UbcH8, but not with UbcH5 and UbcH7, suggesting that UbcH8 is a cognate E2 enzyme for Siah-1.

View larger version (29K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 3. Siah-1 binds UbcH8 and promotes ubiquitination but not degradation of -synuclein. (A) Siah-1 interacts specifically with UbcH8. HeLa cells were co-transfected with pMyc-Siah-1 and pCHA-UbcH5, UbcH7 or UbcH8. Lysates (Input) were immunoprecipitated with anti-Myc antibody, followed by immunoblotting with anti-HA and anti-Myc antibodies. (B) Siah-1 promotes mono- and di-ubiquitination of -synuclein. HeLa cells expressing the indicated FLAG-, HA- and Myc-tagged proteins were incubated in the presence or absence of 5 µM MG132 for 24 h. Lysates were immunoprecipitated with anti-FLAG antibody, followed by immunoblotting with anti-Myc (upper panel) and anti--synuclein antibodies (lower panel). The asterisk indicates non-specific bands. (C) Siah-1 overexpression has no effect on -synuclein degradation. HeLa cells co-transfected with pMyc--synuclein and pCHA-Siah-1 or pCHA vector were treated with 20 µM MG132 or vehicle for 8 h. Cell lysates were analyzed by immunoblotting with anti-Myc (top), anti-HA (middle) or anti-actin (bottom) antibodies. The asterisk indicates a degradation product of HA-Siah-1.

 
Our finding of a specific interaction and colocalization between Siah-1 and -synuclein (Figs 1 and 2) raises the possibility that Siah-1 may ubiquitinate -synuclein. To test this possibility, we used a well established in vivo ubiquitination assay (36–41) to examine the effects of Siah-1 overexpression on -synuclein ubiquitination in transfected HeLa cells (Fig. 3B). We found that, in the absence of exogenous Siah-1, -synuclein was ubiquitinated, as revealed by the presence of Myc-tagged ubiquitin on -synuclein. The ubiquitination of -synuclein was significantly enhanced by co-expression of Siah-1, supporting a role for Siah-1 in ubiquitinating -synuclein. Only mono- and di-ubiquitinated -synuclein species but not polyubiquitinated forms of -synuclein were detected, indicating that Siah-1 facilitates mono- and di-ubiquitination rather than polyubiquitination of -synuclein. The levels of mono- and di-ubiquitination of -synuclein were increased by treatment with the proteasome inhibitor MG132 (Fig. 3B), which is likely due to the increased Siah-1 protein expression caused by proteasome inhibition (Fig. 3C) (26,42,43).

Siah-1-mediated ubiquitination has no significant effect on the degradation of -synuclein
Given the previous reports that Siah-1 promotes the degradation of its substrates by the ubiquitin–proteasome pathway (35,42,44), we sought to determine whether Siah-1 has a role in regulating -synuclein degradation. HeLa cells co-transfected with pMyc--synuclein and pCHA-Siah-1 or pCHA vector were treated for 8 h with MG132 or vehicle, and the levels of -synuclein and Siah-1 were then analyzed by western blotting. As previously reported (42,43), the Siah-1 level was increased upon inhibition of the proteasome function by MG132 (Fig. 3C), indicating that the stability of Siah-1 is controlled by the proteasome-mediated degradation. Overexpression of Siah-1 and MG132 treatment had no significant effect on the -synuclein level, suggesting that, unlike other Siah-1 substrates, -synuclein ubiquitinated by Siah-1 is not targeted for degradation by the proteasome. These results are consistent with our finding (Fig. 3B) that the types of ubiquitination of -synuclein mediated by Siah-1 are mono- and di-ubiquitination, which have a non-degradative role in cells (45).

Ubiquitination of -synuclein by Siah-1 is abolished by A30P but not A53T -synuclein mutation
Next, we investigated whether Siah-1-mediated ubiquitination of -synuclein is affected by PD-linked A30P and A53T -synuclein mutations. We first performed co-immunoprecipitation experiments to determine the effects of A30P and A53T mutations on the interaction of -synuclein with Siah-1. We found that both A30P and A53T -synuclein mutants were capable of interacting with Siah-1, although their ability to bind Siah-1 were significantly reduced compared with that of wild-type -synuclein (Fig. 4A and B). We then performed the same in vivo ubiquitination assays as described for wild-type (WT) -synuclein (Fig. 3B) to examine the effects of A30P and A53T mutations on the ubiquitination of -synuclein by Siah-1. We found that the mono- and di-ubiquitination of -synuclein by Siah-1 was completely abolished by A30P mutation but not by A53T mutation (Fig. 4C). Like wild-type -synuclein (Fig. 3B), A53T -synuclein mutant protein exhibited increased mono- and di-ubiquitination by Siah-1 in response to proteasome inhibition by MG132 (Fig. 4C), likely as a result of the proteasome inhibition-induced increase in Siah-1 protein expression (Fig. 3C) (26,42,43). We found that overexpression of Siah-1 and MG132 treatment had no significant effect on the protein levels of A30P and A53T -synuclein mutants (Fig. 4C), suggesting that these -synuclein mutants are not degraded by the ubiquitin–proteasome pathway.

View larger version (32K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 4. Effects of PD-linked -synuclein mutations on ubiquitination of -synuclein by Siah-1. (A) Effects of A30P and A53T mutations on the interaction of -synuclein with Siah-1. HeLa cells were co-transfected with pMyc-Siah-1 and pCHA-WT, A30P or A53T -synuclein. Lysates were immunoprecipitated with an anti-HA antibody, followed by immunoblotting with anti-Myc and anti-HA antibodies. (B) The level of Siah-1 binding to WT or mutant -synuclein is quantified from co-immunoprecipitation experiments described in (A) and normalized to the immunoprecipitated -synuclein level in each sample, and is expressed as a percentage of Siah-1 binding to WT -synuclein. Data represent mean±SEM from three independent experiments. *Significantly different from the level of Siah-1 binding to WT -synuclein (P < 0.05). (C) Effects of A30P and A53T mutations on the ubiquitination of -synuclein by Siah-1. HeLa cells expressing the indicated FLAG-, HA- and Myc-tagged proteins were incubated in the presence or absence of 5 µM MG132 for 24 h. Lysates were immunoprecipitated with anti-HA antibody followed by immunoblotting with anti-Myc and anti-HA antibodies.

 
Siah-1-mediated ubiquitination increases the insolubility of -synuclein
Given our findings that Siah-1 facilitates mono- and di-ubiquitination of -synuclein and does not alter the degradation of -synuclein (Figs 3 and 4), we next investigated whether Siah-1-mediated ubiquitination of -synuclein affects the solubility of -synuclein. HA-tagged wild-type, A30P or A53T mutant -synuclein was co-expressed in HeLa cells with Myc-tagged ubiquitin in the absence or presence of FLAG-tagged Siah-1. Cells were treated for 24 h with MG132 or vehicle, and cell lysates were separated into Triton X-100 (TX)-soluble and -insoluble fractions to assess the TX solubility of non-ubiquitinated and ubiquitinated forms of wild-type and mutant -synuclein (Fig. 5). We found that the non-ubiquitinated wild-type, A30P and A53T mutant -synuclein were distributed in both TX-soluble and -insoluble fractions. In contrast, the ubiquitinated wild-type and A53T mutant -synuclein proteins were only present in the TX-insoluble fraction but not in the TX-soluble fraction (Fig. 5). Consistent with our in vivo ubiquitination results (Figs 3B and 4C), we found that A30P mutant -synuclein was not ubiquitinated and that MG132 treatment and Siah-1 overexpression significantly increased the amount of mono- and di-ubiquitinated forms of wild-type and A53T mutant -synuclein in the TX-insoluble fraction without affecting the total protein levels of wild-type or mutant -synuclein. Together, these data provide evidence that Siah-1-mediated ubiquitination increases the insolubility of -synuclein.

View larger version (29K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 5. Ubiquitination of -synuclein by Siah-1 increases the insolubility of -synuclein. HeLa cells expressing the indicated FLAG-, HA- and Myc-tagged proteins were treated with 5 µM MG132 or vehicle for 24 h, and cell lysates were separated into Triton X-100 (TX)-soluble and -insoluble fractions. The TX-insoluble pellets were then extracted by a 2% SDS buffer. Both the TX-soluble and SDS-soluble fractions were subjected to immunoprecipitation with anti-HA antibody, followed by immunoblotting with anti-Myc (top panels) and anti-HA (bottom panels) antibodies.

 
Siah-1-mediated ubiquitination promotes the formation of -synuclein-positive inclusion bodies
-Synuclein has been shown to aggregate into cytoplasmic inclusion bodies in response to impairment of proteasome function by proteasome inhibitors (46,47). To determine whether the increased insolubility of -synuclein induced by Siah-1-mediated ubiquitination is associated with an increased propensity of -synuclein to form intracellular aggregates, we assessed the effects of Siah-1 overexpression on MG132-induced -synuclein aggregation in PC12 cells expressing HA-tagged wild-type, A30P or A53T -synuclein (Fig. 6). Immunostaining of MG132-treated PC12 cells with anti-HA antibodies revealed the presence of prominent -synuclein aggregates, which accumulate in the perinuclear area (Fig. 6A). The -synuclein aggregates were also stained positive for Thioflavin S (Thio S), a fluorescent dye which labels β-sheet-rich inclusion bodies, including Lewy bodies (48,49). Quantification of both HA--synuclein- and Thio S-positive inclusion bodies showed that (Fig. 6B), in cells overexpressing wild-type -synuclein, co-expression of Siah-1 significantly increased the percentage of cells with inclusions (39.6 ± 1.4%, n = 3; P < 0.05) compared with the vector-transfected controls (23.7 ± 0.5%, n = 3). Co-expression of Siah-1 in cells overexpressing A53T -synuclein also resulted in a similar extent of increase in the formation of HA--synuclein- and Thio S-positive inclusion bodies (37.5 ± 0.9%, n = 3; P < 0.05) compared to the vector–transfected cells overexpressing A53T -synuclein (26.6 ± 3.0%, n = 3). In contrast, co-expression of Siah-1 in cells overexpressing A30P -synuclein had no significant effect on inclusion body formation (31.7 ± 2.5%, n = 3) compared with the vector-transfected cells overexpressing A30P -synuclein (33.9 ± 4.7%, n = 3). These results correlate well with the ability of wild-type or mutant -synuclein to serve as a Siah-1 substrate (Figs 3B and 4B) and thus provide strong support for a direct link between Siah-1-mediated -synuclein ubiquitination and the formation of -synuclein-positive inclusion bodies.

View larger version (52K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 6. Siah-1 promotes aggregation of WT and A53T but not A30P -synuclein into inclusion bodies. (A) Representative images of PC12 cells co-transfected with pCHA--synuclein and pMyc vector (upper panel) or pMyc-Siah-1 (lower panel), treated with 5 µM MG132 for 24 h, and then stained with Thio S (green), DAPI (blue) and anti-HA antibody (red). Scale bar=10 µm. (B) The percentage of transfected cells containing inclusion bodies positive for both -synuclein and Thio S was quantified for each transfection group as indicated. Data represent mean±SEM from three independent experiments. *Significantly different from the corresponding Myc vector-transfected control cells (P < 0.05). Thio S, Thioflavin S; DAPI, 4',6-Diamidino-2-phenylindole.

 
Siah-1-mediated -synuclein ubiquitination enhances apoptotic cell death
Next, we investigated whether Siah-1-mediated -synuclein ubiquitination has a role in cell vulnerability to proteasome impairment. PC12 cells were co-transfected with pCHA--synuclein (WT, A30P or A53T) or pCHA vector and pMyc-Siah-1 or pMyc vector, treated with MG132, and cell viability was assessed using the MTT assay (Fig. 7A). For cells transfected with the HA and Myc vectors, exposure to 5 µM MG132 caused cell viability to be decreased to 79.8 ± 2.4% of vehicle-treated controls (n = 3) and overexpression of Siah-1 had no significant effect on cell viability. In contrast, for cells overexpressing wild-type -synuclein, Siah-1 overexpression significantly decreased cell viability (53.2 ± 0.5%, n = 3; P < 0.05) compared with the Myc vector-transfected controls (70.3 ± 1.4%, n = 3). A similar reduction in cell viability by Siah-1 overexpression was also observed in cells overexpressing A53T -synuclein (57.8 ± 1.2%, n = 3; P < 0.05) compared with the corresponding Myc vector-transfected controls (66.7 ± 1.1% n = 3). However, in cells overexpressing A30P -synuclein, Siah-1 overexpression did not significantly change cell viability (63.4 ± 1.7%; n = 3) compared with the corresponding Myc vector-transfected control cells (67.1 ± 0.8%; n = 3). These data, together with the results of wild-type, A30P and A53T -synuclein ubiquitination by Siah-1 (Figs 3B and 4B), suggest that Siah-1-mediated -synuclein ubiquitination makes cells more vulnerable to proteasome impairment.

View larger version (35K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 7. Siah-1 enhances apoptotic cell death in cells overexpressing WT and A53T but not A30P -synuclein. (A) PC12 cells co-transfected with the indicated pCHA--synuclein (WT, A30P or A53T) or pCHA vector and pMyc-Siah-1 or pMyc vector were treated with 5 µM MG132 or vehicle for 24 h. The extent of cell survival was assessed by using the MTT assay and is expressed as the percentage of cell viability in the corresponding vehicle-treated control cells. Data represent mean±SEM from three independent experiments. *Significantly different from the corresponding Myc vector-transfected control cells (P < 0.05). (B) Immunoblot analysis of cytochrome c (Cyt. c) and actin in the cytosolic fractions isolated from MG132-treated cells co-transfected with the indicated HA- and Myc-tagged constructs. The level of cytochrome c released to the cytosol is normalized to the level of actin in each cell sample. Data represent mean±SEM from three independent experiments. *Significantly different from the corresponding Myc vector-transfected control cells (P < 0.05). AU, arbitrary units.

 
To provide additional evidence supporting an association between Siah-1-mediated -synuclein ubiquitination and cell vulnerability to proteasome impairment, we performed cytochrome c release assays (50) to examine the effects of Siah-1 overexpression on MG132-induced apoptotic cell death in cells overexpressing wild-type, A30P, or A53T -synuclein, or vector-transfected controls (Fig. 7B). We found that, in cells overexpressing WT or A53T -synuclein, Siah-1 overexpression caused a significant increase in the MG132-induced release of cytochrome c from mitochondria to the cytosol compared with the corresponding Myc vector-transfected controls. In contrast, Siah-1 overexpression had no apparent effect on MG132-induced release of cytochrome c from cells overexpressing A30P -synuclein or from HA vector-transfected control cells. These data are consistent with the effects of Siah-1 overexpression on cell viability and suggest that Siah-1-mediated -synuclein ubiquitination promotes apoptotic cell death.

Siah-1 depletion decreases -synuclein ubiquitination and rescues cytotoxicity induced by -synuclein overexpression and proteasome impairment
To investigate the role of endogenous Siah-1 in the regulation of -synuclein ubiquitination, we examined the effect of small interfering RNA (siRNA)-mediated Siah-1 knockdown on the ubiquitination of endogenous -synuclein in PC12 cells. As shown in Fig. 8A, Siah-1 siRNA-1 and Siah-1 siRNA-2, two distinct siRNA duplexes targeting different regions of Siah-1 mRNA, both specifically inhibited the expression of endogenous Siah-1 in PC12 cells, although Siah-1 siRNA-2 caused a greater decrease in Siah-1 protein level than Siah-1 siRNA-1. In vivo ubiquitination analysis revealed that, in PC12 cells transfected with Siah-1 siRNA-2, ubiquitination of endogenous -synuclein was greatly decreased compared with NT siRNA-treated control cells (Fig. 8B). Conversely, ubiquitination of endogenous -synuclein was significantly increased by Siah-1 overexpression in PC12 cells (Fig. 8B). These results further confirm that -synuclein is indeed a physiological substrate for Siah-1 E3 ligase.

View larger version (23K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 8. Siah-1 knockdown reduces -synuclein ubiquitination and MG132-induced cell death in cells overexpressing WT and A53T but not A30P -synuclein. (A) PC12 cells were transfected with non-targeting (NT) siRNA, Siah-1 siRNA-1 or Siah-1 siRNA-2. The levels of Siah-1 and actin in the cell lysates were analyzed by immunoblotting with anti-Siah-1 and anti-actin antibodies. (B) PC12 cells transfected with pRK5-HA-ubiquitin alone (control) or in combination with Siah-1 siRNA-2 or pMyc-Siah-1 were treated with 5 µM MG132 for 24 h. Ubiquitination of endogenous -synuclein was analyzed by immunoprecipitation with anti--synuclein antibody, followed by immunoblotting with anti-HA (upper panel) and anti--synuclein (lower panel) antibodies. (C) PC12 cells co-transfected with indicated HA-tagged -synuclein constructs and Siah-1 siRNA-2 treated with 50 µM MG132 for 24 h. The extent of cell survival was assessed by using the MTT assay and is expressed as the percentage of cell viability in the corresponding vehicle-treated control cells. Data represent mean±SEM from three independent experiments. *Significantly different from the corresponding NT siRNA-transfected control cells (P < 0.05).

 
Next, we examined the effects of Siah-1 depletion on MG132-induced cell death in PC12 cells overexpressing wild-type, A30P or A53T -synuclein or vector-transfected controls (Fig. 8C). We found that, in cells overexpressing wild-type -synuclein, Siah-1 depletion significantly reduced cell death induced by exposure to 50 µM MG132; Siah-1 depleted cells had increased cell viability (56.6 ± 0.2%, n = 3; P<0.05) compared with the NT siRNA-treated controls (41.6 ± 1.3%; n =3). A similar increase in cell viability by Siah-1 depletion was also observed in cells overexpressing A53T -synuclein (45.4 ± 0.9%, n = 3; P < 0.05) compared with the corresponding NT siRNA-treated control cells (38.8 ± 0.7%; n = 3). In contrast, in cells overexpressing A30P -synuclein, Siah-1 depletion had no significant effect on cell viability (39.1 ± 1.6%; n = 3) compared with the corresponding NT siRNA-treated control cells (39.0 ± 1.2%; n = 3). Together, these data suggest that Siah-1-mediated -synuclein ubiquitination plays a critical role in regulation of -synuclein toxicity under conditions in which the proteasome function is impaired.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS FUNDING REFERENCES

 
While accumulating evidence indicates that -synuclein is ubiquitinated in cells (51) and in Lewy bodies (14,16,17), little is known about the functional significance of -synuclein ubiquitination and the molecular mechanisms that regulate -synuclein ubiquitination. The present study demonstrates that -synuclein binds and colocalizes with Siah-1 E3 ligase in cells. These results are consistent with the presence of a VXP motif (residues 118–120) in -synuclein, which fits the core consensus sequence for binding to the substrate-recognition site of Siah-1 (52,53). Our biochemical data indicate that Siah-1 binds brain-enriched ubiquitin-conjugating enzyme UbcH8, although it remains to be determined whether UbcH8 actually participates in Siah-1-mediated ubiquitination reaction.

Our results of in vivo ubiquitination assays using epitope-tagged ubiquitin reveal that Siah-1 E3 ligase facilitates mono- and di-ubiquitination of -synuclein. Unfortunately, despite repeated attempts, we were unable to get a good signal when we probed the immunoprecipitated -synuclein with anti-ubiquitin antibodies. However, the -synuclein ubiquitination detected with epitope-tagged ubiquitin is likely to be physiologically relevant for the following reasons: (i) The use of epitope-tagged ubiquitin is a well-established method that has been widely used for detection of protein ubiquitination (36–41). Given the high abundance of ubiquitin in cells, expression of epitope-tagged ubiquitin is believed to have no effect on ubiquitination states of proteins (36–38). (ii) Our observed mono- and di-ubiquitination of -synuclein is similar to the previously reported ubiquitination pattern of -synuclein (14–17,51). The results that -synuclein ubiquitination is increased by Siah-1 overexpression and decreased by Siah-1 knockdown indicate that the observed ubiquitination of -synuclein is a specific effect that is dependent on Siah-1 levels rather than a non-specific consequence of expressing epitope-tagged ubiquitin.

In our studies, we observed that the di-ubiquitinated -synuclein sometimes appeared as a doublet (Fig. 3B), whereas at other times appeared as a single band (Figs 4B and 8B). Such variable appearance of the di-ubiquitinated -synuclein doublet is also apparent in previous studies of -synuclein ubiquitination (14–17). The reason for this variability is unclear, but the di-ubiquitinated -synuclein doublet might be caused by phosphorylation (16,17) or proteolysis (17) of -synuclein. Interestingly, the Siah-1-mediated mono- and di-ubiquitination of -synuclein is significantly increased in response to proteasome inhibition, which is likely due to an increase in Siah-1 protein level induced by proteasome impairment (26,42,43). Our findings that the ubiquitination of endogenous -synuclein is greatly enhanced by Siah-1 overexpression and dramatically reduced by siRNA-mediated depletion of Siah-1 provide strong evidence that Siah-1 is a major E3 ligase responsible for mono- and di-ubiquitination of endogenous -synuclein in cells.

Ubiquitination involves formation of an isopeptide bond between the C-terminal glycine residue of ubiquitin and the -amino group of a lysine residue on the substrate (54,55). Among the total of 15 lysine residues present in -synuclein protein sequence, four residues (K21, K23, K32 and K34) were reported to be ubiquitinated in vitro by rabbit reticulocyte lysates (51) and three residues (K12, K21 and K23) were found to be ubiquitinated in Lewy bodies (17). Although Siah-1-mediated -synuclein ubiquitination sites remain to be identified, our results that ubiquitination of -synuclein by Siah-1 is abolished by PD-linked A30P but not A53T -synuclein mutation suggest that some of these -synuclein lysine residues (e.g. K21, K23, K32 and K34) near Ala-30 may be the ubiquitination sites by Siah-1 E3 ligase and their ubiquitination could be inhibited by A30P-induced conformational change in -synuclein.

Ubiquitination is a dynamic post-translational modification that serves diverse cellular roles (54,55). While K48-linked polyubiquitination acts as the canonical signal for targeting protein to the proteasome for degradation, mono-ubiquitination and other types of ubiquitination function in a manner analogous to phosphorylation for modulating protein activity, location and interactions (41,56). We and others have previously shown that Siah-1 facilitates polyubiquitination and subsequent proteasomal degradation of its substrates, such as synaptophysin (26) and synphilin-1 (35). In contrast, our current study reveals that Siah-1 promotes mono- and di-ubiquitination of -synuclein and does not regulate the degradation of -synuclein. Our observation that proteasome inhibition by MG132 has no significant effect on the protein levels of wild-type, A30P and A53T mutant -synuclein suggests that most wild-type and mutant -synuclein proteins are not degraded by the proteasome, in agreement with recent reports (57,58).

Ample evidence indicates that -synuclein is a natively unfolded protein that is prone to aggregation (59–61). Recombinant -synuclein has been shown to undergo spontaneous aggregation into fibrils in vitro (49), and the propensity of -synuclein to form fibrils is significantly increased by PD-linked A30P and A53T mutation, although these two mutant -synucleins differ in their rates of fibril formation (62). Previous studies have shown the presence of non-ubiquitinated as well as mono- and di-ubiquitinated forms of -synuclein in the TX-insoluble fractions from PD brains (14–17,51). Consistent with these reports, our biochemical data show that a substantial amount of the non-ubiquitinated form of wild-type, A30P and A53T mutant -synuclein is present in the TX-insoluble fraction from transfected cells. Furthermore, A30P -synuclein, a mutant form of -synuclein, which is not ubiquitinated by Siah-1, is able to form intracellular aggregates. These results indicate that ubiquitination of -synuclein is not required for -synuclein aggregation. Our analyses of the effects of mono- and di-ubiquitination of wild-type and A53T mutant -synuclein by Siah-1 reveal that ubiquitination of -synuclein enhances the insolubility of -synuclein and increases the propensity of -synuclein to form intracellular aggregates. Taken together, our results suggest that, although Siah-1-mediated mono- and di-ubiquitination of -synuclein is not required for -synuclein aggregation, the ubiquitination of -synuclein by Siah-1 has a regulatory role in promoting -synuclein aggregation.

While our data clearly show that the ubiquitinated forms of -synuclein are in the TX-insoluble fraction, it remains unknown whether ubiquitination of -synuclein occurs prior to or after -synuclein aggregation. A previous study reported that the un-aggregated, monomeric form and aggregated, fibrillar form of -synuclein can both undergo mono- and di-ubiquitination in vitro (15), suggesting that ubiquitination of -synuclein can take place either prior to or after -synuclein aggregation. It has become increasingly clear that -synuclein aggregation is a complex multi-step process that results in several different kinds of intermediates and products, including small, soluble oligomers and highly ordered, β-sheet-rich fibrils (63,64). Recent evidence suggests that small oligomers or protofibrils may be the principal toxic species responsible for neuronal cell death (64). Future studies are required to determine where ubiquitination of -synuclein occurs along the -synuclein aggregation pathway and how it affects -synuclein aggregation process.

Although whether -synuclein aggregates are cytotoxic or cytoprotective remains a hotly debated issue, the presence of -synuclein aggregates in PD and other -synucleinopathies suggests that the -synuclein aggregates per se, or some event associated with -synuclein aggregation process, is toxic to neurons. The recent identification of -synuclein gene duplication and triplication as a cause for familial PD (6–8) provide direct evidence linking increased levels of wild-type -synuclein to neurodegeneration in PD. Our data indicate that mono- and di-ubiquitination of -synuclein by Siah-1 not only enhance -synuclein aggregation, but also promote apoptotic cell death induced by -synuclein overexpression and proteasome inhibition. Our findings reveal a critical role for Siah-1-mediated mono- and di-ubiquitination of -synuclein in regulation of -synuclein aggregation and toxicity.

Proteasome dysfunction and -synuclein aggregation have both been strongly implicated in PD pathogenesis (65,66). It is well established that Lewy bodies and other -synuclein-containing inclusions in -synucleinopathies are immunoreactive to anti-ubiquitin antibodies (12,13), and recent evidence indicates that -synuclein is the major ubiquitinated protein in Lewy bodies (17). However, the relationship between proteasome impairment and ubiquitination/aggregation of -synuclein remains unclear. Our data obtained from the present study support a model that, under the pathogenic conditions in which proteasome function is impaired, the level of Siah-1 E3 ligase is dramatically increased, leading to enhanced mono- and di-ubiquitination of -synuclein and thereby promotes -synuclein aggregation and toxicity. Our results suggest Siah-1-mediated -synuclein ubiquitination as a potential target for therapeutic intervention in PD.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS FUNDING REFERENCES

 
Plasmids and antibodies
Conventional molecular biological techniques were used to generate the following expression constructs with N-terminal HA (pCHA), Myc (pMyc) or FLAG (pFLAG) tags: Siah-1, WT--synuclein, A30P--synuclein and A53T--synuclein (26). Antibodies used in this study include: anti-Siah-1 N-15 and anti-ubiquitin FL76 (Santa Cruz Biotechnology, Inc.); anti--synuclein and anti-cytochrome c 7H8 (BD Transduction Laboratories); anti-HA 3F10 (Roche Molecular Biological Sciences); anti-ubiquitin P4G7 and anti-HA.11 (Covance); anti-FLAG M2 (Sigma); anti-Myc 9E10.3 (Neomarkers); and anti-actin C4 (Chemicon). All secondary antibodies were purchased from Jackson ImmunoResearch Laboratories, Inc.

GST pull-down assays
The full-length rat Siah-1 cDNA was subcloned into a pGEX-5X-2 vector (GE Healthcare) to obtain GST-Siah-1 fusion proteins as previously described (26). GST-Siah-1 fusion proteins and GST protein controls were immobilized on glutathione-agarose beads (Sigma) and incubated with rat brain homogenates for 2 h at 4°C in 50 mM NaH₂PO₄ (pH 7.2) and 0.05% Triton X-100 (26). After extensive washes, bound proteins were eluted by boiling in Laemmli sample buffer, and analyzed by SDS–PAGE and immunoblotting. Antibody binding was visualized using the enhanced chemiluminescence system (GE Healthcare) (67).

Cell transfections and immunoprecipitations
HeLa and PC12 cells were transfected with the indicated plasmids using Lipofectamine 2000 reagent (Invitrogen) and TransIT-Neural Reagent (Mirus), respectively, according to the manufacturer's instructions. Immunoprecipitations were carried out as described previously (26,68) using cell lysates with anti-HA, anti-Myc, anti-FLAG or anti--synuclein antibodies. Immunocomplexes were analyzed by SDS–PAGE and immunoblotting (67).

siRNAs transfection
For depletion of Siah-1 in PC12 cells, siRNAs (Qiagen) were generated against the following rat Siah-1 mRNA sequences: Siah-1 siRNA-1, 5'-GAUAGGAACACGCAAGCAA-3'; and Siah-1 siRNA-2, 5'-GUUGCAUGUAGUAACACUA-3'. A control siRNA (NT siRNA) with no known mammalian homology (siCONTROL Non-Targeting siRNA #1, Dharmacon) was used as a negative control. PC12 cells were transfected with the indicated siRNAs using the TransIT siQuest reagent (Mirus) as described previously (68). Experiments were performed 72 h after siRNA treatment.

Immunofluorescence confocal microscopy
Primary cortical cultures were prepared from embryonic day 18 mouse brains as described (69) and maintained in NeuroBasal Media (Gibco) supplemented with AraC (Sigma) for 7–14 days. PC12 cells were differentiated with nerve growth factor (NGF, 50 ng/ml) for 72 h (68). Cells were fixed in 4% paraformaldehyde and processed for indirect immunofluorescence microscopy as previously described (26). The staining patterns were examined using a Zeiss LSM 510 confocal microscope and images processed using Adobe Photoshop 7.0 (Adobe Systems, Inc.).

Analysis of Siah-1 effect on steady-state -synuclein levels
HeLa cells expressing Myc--synuclein and HA-Siah-1 or HA vector were incubated for 8 h at 37°C with the proteasome inhibitor MG132 (Sigma) (20 µM) or vehicle (DMSO, final concentration 0.1%) as previously described (26). Cells were then lysed, and an equal amount of protein from each lysate was analyzed by SDS–PAGE followed by immunoblotting for Myc, HA and actin.

In vivo ubiquitination assays
In vivo ubiquitination assays were performed as described previously (70). HeLa cells were co-transfected with pMyc-Ubiquitin and the indicated Siah-1 and -synuclein expression constructs. Twenty-four hours post-transfection, cells were incubated for 24 h with 5 µM MG132 or vehicle (DMSO, final concentration 0.1%). The cells were then lysed, and an equal amount of protein from each lysate was immunoprecipitated using anti-FLAG or anti-HA antibodies as indicated. Immunocomplexes were analyzed by SDS–PAGE followed by immunoblotting with an anti-Myc antibody to detect ubiquitin conjugates. For analysis of ubiquitination of endogenous -synuclein in PC12 cells, cells were transfected with pRK5-HA-Ubiquitin and either pMyc-Siah-1 or Siah-1 siRNA. Twenty-four hours post-transfection, cells were incubated with 5 µM MG132 for 24 h. Cell lysates were immunoprecipitated with anti--synuclein antibody, followed by immunoblotting with anti-HA antibody to detect ubiquitin conjugates.

Cell fractionation
Transfected HeLa cells were lysed in buffer containing 50 mM Tris–HCl (pH 7.6), 150 mM NaCl, 1.0% Triton X-100 and protease inhibitors (71). Lysates were centrifuged at 100 000 g for 30 min at 4°C and separated into Triton X-100 soluble (supernatant) and insoluble (pellet) fractionations. The Triton X-100 insoluble pellets were resuspended in boiling 2% SDS by sonication and then diluted 3x in immunoprecipitation buffer (72). Immunoprecipitations of Triton X-100 soluble and insoluble fractionations were performed as described previously (67).

Analysis of inclusion formation
PC12 cells co-transfected with pCHA-WT, pCHA-A30P or pCHA-A53T -synuclein and pMyc-Siah-1 or pMyc3L vector were treated with 5 µM MG132 for 24 h at 37°C. Cells were then incubated with 0.01% Thioflavin S (Sigma) for 8 min (73) and processed for subsequent immunostaining with anti-HA antibody (26). Stained cells were visualized using a Lietz DMRBE fluorescence light microscope (Leica). The number of transfected cells with inclusions positive for both -synuclein and Thioflavin S were counted in 5–6 randomly selected fields from each transfection group by an investigator in a blinded manner. Experiments were repeated three times, and the data were subjected to statistical analysis by student t-test.

Cell viability and cytochrome c release assays
Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay as described (74). The cytochrome c release assay was performed as we described previously (50). Briefly, transfected cells were homogenized and then fractionated into the mitochondria and cytosol fractions. The release of cytochrome c to the cytosol was determined by immunoblotting with anti-cytochrome c antibody.

	FUNDING

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS FUNDING REFERENCES

 
This work was supported by grants from National Institutes of Health (AG021489, NS050650 and NS047199).

	ACKNOWLEDGEMENTS

 
We thank Dr Ted Dawson for providing cDNAs for HA-tagged E2 enzymes (UbcH5, UbcH7 and UbcH8) and Elizabeth Webber for her critical reading of the manuscript.

Conflict of Interest statement. None declared.

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