Dorfin Localizes to Lewy Bodies and Ubiquitylates Synphilin-1*

Takashi Ito, Jun-ichi Niwa {ddagger}, Nozomi Hishikawa, Shinsuke Ishigaki, Manabu Doyu and Gen Sobue §

From the Department of Neurology, Nagoya University Graduate School of Medicine, Showa-ku, Nagoya 466-8550, Japan

Received for publication, March 18, 2003 , and in revised form, May 12, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Parkinson's disease (PD) is a neurodegenerative disease characterized by loss of nigra dopaminergic neurons. Lewy bodies (LBs) are a characteristic neuronal inclusion in PD brains. In this study, we report that Dorfin, a RING finger-type ubiquityl ligase for mutant superoxide dismutase-1, was localized with ubiquitin in LBs. Recently, synphilin-1 was identified to associate with {alpha}-synuclein and to be a major component of LBs. We found that overexpression of synphilin-1 in cultured cells led to the formation of large juxtanuclear inclusions, but showed no cytotoxicity. Dorfin colocalized in these large inclusions with ubiquitin and proteasomal components. In contrast to full-length synphilin-1, overexpression of the central portion of synphilin-1, including ankyrin-like repeats, a coiled-coil domain, and an ATP/GTP-binding domain, predominantly led to the formation of small punctate aggregates scattered throughout the cytoplasm and showed cytotoxic effects. Dorfin and ubiquitin did not localize in these small aggregates. Overexpression of the N or C terminus of synphilin-1 did not lead to the formation of any aggregates. Dorfin physically bound and ubiquitylated synphilin-1 through its central portion, but did not ubiquitylate wild-type or mutant {alpha}-synuclein. These results suggest that the central domain of synphilin-1 has an important role in the formation of aggregates and cytotoxicity and that Dorfin may be involved in the pathogenic process of PD and LB formation by ubiquitylation of synphilin-1.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Parkinson's disease (PD)1 is a neurodegenerative disease caused by loss of nigra dopaminergic neurons. Lewy bodies (LBs) are a characteristic neuronal inclusion in PD brains (14). Although LBs are a prominent pathological feature of PD, the underlying molecular mechanism accounting for LB formation is poorly understood. Several lines of evidence have suggested that derangements in the ubiquitin/proteasome protein degradation pathway play a prominent role in the pathogenesis of PD (5). Ubiquitin and proteasome subunits colocalize in LBs (6, 7), and biochemical studies have revealed reduced catalytic activities of proteasomes in the lesions of PD (8, 9). The gene product responsible for autosomal recessive juvenile parkinsonism, parkin (10), is an E3 ubiquityl ligase (1113). Accumulation of target protein(s) due to loss of the ubiquitylation function of parkin may contribute to the development of autosomal recessive juvenile parkinsonism. In addition, a missense mutation in UCHL1 (ubiquitin C-terminal hydrolase L1) has been described in a family with PD (14). UCHL1 produces monomeric ubiquitin by cleaving polyubiquitin chains (15). Recently, ubiquityl ligase activity as well as the hydrolase activity of UCHL1 were also reported (16).

{alpha}-Synuclein is a 19-kDa presynaptic vesicular protein of unconfirmed function and one of the major components of LBs (17, 18). Mutations in {alpha}-synuclein (A30P and A53T) cause a rare autosomal dominant form of PD, which shares many phenotypic characteristics with sporadic PD (19, 20). {alpha}-Synuclein aggregates deposit in LBs in both autosomal dominant and sporadic forms of PD (21, 22). In addition, it has been reported that transgenic flies and mice overexpressing human wild-type or mutant {alpha}-synuclein have abnormal cellular accumulation of {alpha}-synuclein and neuronal dysfunction and degeneration (2330), indicating that {alpha}-synuclein has a role in the pathogenesis of both familial and sporadic forms of PD.

Synphilin-1 was identified recently by yeast two-hybrid techniques as a novel protein that interacts with {alpha}-synuclein (31). {alpha}-Synuclein amino acids 1–65 are is sufficient for interaction, and the central portion of synphilin-1 (amino acids 349–555) is necessary and sufficient for interaction with {alpha}-synuclein (32). It has also been reported that the C terminus of {alpha}-synuclein is closely associated with the C terminus of synphilin-1 and that a weak interaction occurs between the N terminus of {alpha}-synuclein and synphilin-1 (33). Synphilin-1 is highly concentrated in presynaptic nerve terminals, and its association with synaptic vesicles is modulated by {alpha}-synuclein (34). Coexpression of {alpha}-synuclein and synphilin-1 in transfected cells results in the formation of eosinophil cytoplasmic inclusions that resemble LBs (31, 35), whereas transfection of synphilin-1 alone without expression of {alpha}-synuclein or parkin can also produce cytoplasmic inclusions in cultured cells (36, 37). Furthermore, synphilin-1 is ubiquitylated and degraded by proteasomes in human embryonic kidney 293 (HEK293) cells (37) and is localized as another major component of LBs in the brains of patients with PD (38, 39). Thus, the process through which aggregations are formed by synphilin-1 may be important in the pathogenesis of PD.

Dorfin is a gene product (which we cloned from anterior horn tissues of human spinal cord) (40) that contains a RING finger/IBR (in between RING finger) motif (41) at its N terminus. It was reported that HHARI (human homologue of ariadne) and H7-AP1 (UbcH7-associated protein-1), both RING finger/IBR motif-containing proteins, interact with the ubiquitin carrier protein (E2) UbcH7 through the RING finger/IBR motif and that a distinct subclass of RING finger/IBR motif-containing proteins represents a new family of proteins that specifically interact with distinct E2 enzymes (42, 43). Dorfin is a juxtanuclearly located E3 ubiquityl ligase and may function in the microtubule-organizing centers (40). In the spinal cords of patients with sporadic and familial forms of amyotrophic lateral sclerosis (ALS) with an SOD1 mutation, Dorfin is colocalized with ubiquitin in hyaline inclusions (44). Dorfin physically binds and ubiquitylates various SOD1 mutants derived from familial ALS patients and enhances their degradation (44). Thus, an important and interesting question is whether Dorfin is colocalized with ubiquitin in LBs of PD.

In this study, we show that Dorfin is colocalized with ubiquitin in LBs of PD. We found that Dorfin ubiquitylates synphilin-1 and that overexpression of synphilin-1 leads to ubiquitylated inclusions resembling LBs in cultured cells.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Immunohistochemistry—Immunohistochemical studies were carried out on 20% buffered, Formalin-fixed, paraffin-embedded autopsied brains filed in the Department of Neurology of the Nagoya University Graduate School of Medicine. Five PD brains (67–69 years of age, four men and one woman) and five controls without neurological disease (61–78 years of age, four men and one woman) were studied. The diagnosis of all cases was confirmed by clinical and pathological criteria. Immunohistochemistry was performed as described previously (45). Rabbit polyclonal antiserum was raised against a C-terminal portion of Dorfin (amino acid 678–690) as described previously (40). Dorfin anti-serum (l:200 dilution) and monoclonal anti-ubiquitin antibody (P4D1, 1:400 dilution; Santa Cruz Biotechnology) were used. To assess the colocalization of Dorfin and ubiquitin, a double-labeling immunofluorescence study was performed on selected sections with a combination of anti-Dorfin and anti-ubiquitin antibodies. Anti-Dorfin antibody was visualized by goat anti-rabbit IgG coupled with Alexa Fluor 568 (Molecular Probes, Inc.), and anti-ubiquitin antibody was visualized with sheep anti-mouse IgG coupled with Alexa Fluor 488 (Molecular Probes, Inc.) and observed under a Carl Zeiss LSM-510 laser scanning confocal microscope. For cultured cells, immunostaining was performed as follows. COS-7 cells transiently expressing synphilin-1-DsRed fusion protein in a 4-chamber slide (Nalge Nunc) coated with rat tail collagen (Roche Diagnostics) were fixed with methanol at –20 °C for 10 min, air-dried, and blocked with 5% goat serum for 30 min. Cells were then incubated overnight at 4 °C with the appropriate primary antibody diluted in phosphate-buffered saline. After washing three times with phosphate-buffered saline, Alexa Fluor 488-conjugated secondary antibody (1:1000 dilution; Molecular Probes, Inc.) was added for1hat room temperature. Samples were visualized under an Olympus BX51 epif-luorescence microscope. Primary antibodies against ubiquitin (P4D1, 1:200 dilution), Hsp70 (heat shock protein of 70 kDa; 1:5000 dilution; Stressgen Biotech Corp.), the 20 S proteasome core subunit (1:5000 dilution; Affiniti), and UbcH7 (1:100 dilution; Transduction Laboratories) were used.

Expression Plasmids, Cell Culture, and Transfection—Human synphilin-1 cDNA containing the entire coding region was amplified by Pfu Turbo DNA polymerase (Stratagene) from human brain cDNAs using 5'-GTCAGGATCCACCACCATGGAAGCCCCTGAATACC-3' as the forward primer and 5'-ATATCTCGAGTGCTGCCTTATTCTTTCCTTTG-3' as the reverse primer and inserted in-frame into the BamHI and XhoI sites of the pcDNA3.1/V5His vector (Invitrogen). A plasmid for DsRed-tagged synphilin-1 was constructed by PCR amplification using 5'-ATATCTCGAGACCACCATGGAAGCCCCTGAATACC-3' as the forward primer and 5'-GTCAGGATCCGCCTTTGCCTTATTCTTTCCTTTG-3' as the reverse primer and inserted in-frame into the XhoI and BamHI sites of the pDsRed-N1 vector (Clontech). A series of deletion mutants of synphilin-1 were prepared as synphilin-1-N (amino acid 1–348), synphilin-1-M (amino acid 349–555), and synphilin-1-C (amino acid 556–919). Synphilin-1-M is the central portion of synphilin-1, containing the ankyrin-like repeat, the coiled-coil domain, and the ATP/GTP-binding domain (31). Primers pairs for each deletion mutant were as follows: 5'-GTCAGGATCCACCACCATGGAAGCCCCTGAATACC-3' and 5'-ATATCTCGAGTTCGTCGTGAATTTTGTCT-3' for synphilin-1-N-V5, 5'-ATATCTCGAGACCACCATGGAAGCCCCTGAATACC-3' and 5'-GTCAGGATCCGCTTCGTCGTGAATTTTGTCTAG-3' for synphilin-1-N-DsRed, 5'-GTCAGGATCCACCATGAATGGAAACAATCTAT-3' and 5'-ATATCTCGAGCTTGCCCTCTGATTTCTGG-3' for synphilin-1-M-V5, 5'-ATATCTCGAGACCACCATGAATGGAAACAATCTAT-3' and 5'-GTCAGGATCCGCC TTGCCCTCTGATTTCTGGGC-3 for synphilin-1-M-DsRed, 5'-GTCAGGATCCACCACCATGTCACTCCCTTCTTCAC-3' and 5'-ATATCTCGAGTGCTGCCTTATTCTTTCCTTTG-3' for synphilin-1-C-V5, and 5'-ATATCTCGAGACCACCATGTCACTCCCTTCTTCAC-3' and 5'-GTCAGGATCCGCCTTTGCCTTATTCTTTCCTTTG-3' for synphilin-1-C-DsRed. Construction of pcDNA4/His-Max-Dorfin, pcDNA3.1(+)FLAG-ubiquitin, and pcDNA3.1/MycHis(+)-SOD1 vectors was described elsewhere (40, 44). {alpha}-Synuclein cDNA was amplified by PCR from human brain cDNAs and cloned into the EcoRV site of pcDNA3.1/MycHis(+) (Invitrogen). To generate the mutant {alpha}-synuclein expression vector, A30P and A53T mutations were introduced into pcDNA3.1/MycHis(+)-{alpha}-synuclein with a QuikChange site-directed mutagenesis kit (Stratagene) following the method of Lee et al. (46). COS-7, HEK293, and Neuro2a cells were maintained in Dulbecco's modified Eagle's medium with 10% fetal calf serum. Transfections were performed using the Effectene transfection reagent (QIAGEN Inc.) according to the manufacturer's instructions. To inhibit cellular proteasome activity, cells were treated with 0.5 µM MG132 (carbobenzoxy-L-leucyl-L-leucyl-L-leucinal, Sigma) for 16 h overnight after transfection.

Immunoprecipitation and Western Blot Analysis—Cells were lysed in lysis buffer (50 mM Tris, 150 mM NaCl, 1% Nonidet P-40, and 0.1% SDS) with Complete protease inhibitor mixture (Roche Diagnostics). Immunoprecipitation from transfected cell lysates was performed with 2 µg of antibody and protein A/G Plus-agarose (Santa Cruz Biotechnology), and the immunoprecipitate was then washed four times with lysis buffer. Anti-V5 antibody (Invitrogen) for synphilin-1-V5 fusion proteins and anti-Myc antibody (A-14, Santa Cruz Biotechnology) for {alpha}-synuclein-Myc or SOD1-Myc fusion proteins were used. Immunoprecipitates were subjected to SDS-PAGE and analyzed by Western blotting with ECL detection reagents (Amersham Biosciences).

In Vitro Ubiquitylation Assay—Immunopurified (IP) Xpress-Dorfin bound to anti-Xpress antibody (Invitrogen) with protein A/G Plus-agarose (Santa Cruz Biotechnology) was prepared from lysates of HEK293 cells transfected with pcDNA4/HisMax-Dorfin. IP-synphilin-1-V5 was prepared with anti-V5 antibody bound to protein A/G Plus-agarose from lysates of HEK293 cells transfected with pcDNA3.1/V5His-synphilin-1. IP-{alpha}-synuclein-Myc and IP-SOD1-Myc were prepared with anti-Myc antibodies from lysates of pcDNA3.1/MycHis(+)-{alpha}-synuclein- and pcDNA3.1/MycHis(+)-SOD1-transfected HEK293 cells, respectively. Slurries of IP-Xpress-Dorfin were mixed with IP-synphilin-1-V5, IP-{alpha}-synuclein-Myc, or IP-SOD1-Myc and incubated at 30 °C for 90 min in 50 µl of reaction buffer containing ATP (4 mM ATP in 50 mM Tris-HCl (pH 7.5), 2 mM MgCl2, and 2 mM dithiothreitol), 100 ng of rabbit E1 (Calbiochem), 2 µg of UbcH7 (Affiniti), and 2 µg of His-ubiquitin (Calbiochem). The reaction was terminated by adding 20 µl of 4x sample buffer, and 20-µl aliquots of the reaction mixtures were subjected to SDS-PAGE, followed by Western blotting with anti-His antibody (Novagen).

Neurotoxicity Analysis and Quantification of Synphilin-1 Aggregates—COS-7 cells (1 x 104) were grown overnight on collagen-coated 4-chamber well slides. They were transfected with 0.2 µg of pDsRed-N1-synphilin-1 or its deletion mutants. To inhibit cellular proteasome activity, cells were treated with 0.5 µM MG132 for 16 h overnight after transfection. The number of inclusions was counted in >100 cells randomly selected, and data were averaged from three independent experiments. For cell viability assay, 5 x 103 Neuro2a cells were grown in collagen-coated 96-well plates overnight. They were then transfected with 0.1 µg of pcDNA3.1/V5His-synphilin-1 or deletion mutants of synphilin-1. pcDNA3.1/V5His-LacZ was used as a control. Next, an MTS-based cell proliferation assay was performed using CellTiter 96 (Promega) at 24 h after serum deprivation. The assay was carried out in triplicate. Absorbance at 490 nm was measured in a multiple plate reader.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Dorfin Localizes to LBs of PD—We first examined whether LBs contain Dorfin. Immunohistochemical analysis revealed that Dorfin was predominantly localized in LBs found in PD (Fig. 1A). The peripheral rim of a typical LB in a neuronal cell body was strongly stained, whereas the central core remained unstained (Fig. 1B). Dorfin was also localized in Lewy neurites (Fig. 1C), which are a pathological hallmark in addition to LBs of degenerating neurons in the brains of patients suffering from PD (47). Anti-Dorfin antibody did not stain any abnormal structures in normal brains (data not shown). A double-labeling immunofluorescence study revealed that Dorfin was colocalized with ubiquitin in LBs (Fig. 1, D–F). Serial sections stained with anti-Dorfin and anti-ubiquitin antibodies showed that ~90% of ubiquitylated LBs were positive for Dorfin immunoreactivity. The staining profile of Dorfin was very similar to that of {alpha}-synuclein (48), which is predominantly located in the peripheral rim of LBs, but was different from that of parkin, which localizes predominantly in the core of LBs (49).



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FIG. 1.
Colocalization of Dorfin and ubiquitin in LBs of PD. Substantia nigra tissue of PD cases was immunohistochemically stained with anti-Dorfin antibody. A, LBs (arrows) in neurons were strongly stained. B, the peripheral rim of a typical LB was predominantly stained with anti-Dorfin antibody. C, Lewy neurites (arrowheads) were also Dorfin-immunoreactive. The scale bar in A is equivalent to 100 µm in A and C and 12 µm in B. D–F, shown are laser scanning confocal microscopy images of double-labeling immunofluorescence study of LB. Frozen sections prepared from substantia nigra tissue of PD were incubated with rabbit anti-Dorfin IgG and labeled with Alexa Fluor 568-conjugated anti-rabbit antibodies (red in D) and mouse monoclonal anti-ubiquitin and Alexa Fluor 488-conjugated anti-mouse antibodies (green in E). F shows a merged image of the double-stained LB (D and E), and regions of overlap between Dorfin and ubiquitin immunoreactivities are shown in yellow. The scale bar in D is equivalent to 10 µm and also applies to E and F.

 

Expression of Synphilin-1 Induces LB-like Large Juxtanuclear Inclusions, and Dorfin Localizes to These Inclusions— To investigate the relationships of Dorfin to components of LBs other than ubiquitin, we first examined the subcellular localization of {alpha}-synuclein and synphilin-1 in cultured cells. We created wild-type and mutant {alpha}-synuclein-green fluorescent protein and {alpha}-synuclein-Myc fusion constructs, but there was no evidence of {alpha}-synuclein aggregation in transfected COS-7 cells in the presence or absence of the proteasome inhibitor (data not shown). We created a synphilin-1-DsRed fusion construct by fusing the red fluorescent protein DsRed to the C terminus of synphilin-1 and carried out transient transfection in COS-7 cells with this construct. Large juxtanuclear inclusions were spontaneously formed in the transfected COS-7 cells in the absence of the proteasome inhibitor (Fig. 2, A–O). We also constructed synphilin-1 fusion proteins with a smaller V5/His6 tag, which formed identical inclusions when overexpressed in COS-7 cells, although to a lesser extent than synphilin-1-DsRed fusion proteins (data not shown). Immunostaining with anti-ubiquitin and anti-Dorfin antibodies revealed that most of the large juxtanuclear inclusions of synphilin-1 contained ubiquitin (Fig. 2, A–C) and Dorfin (D–F). Immunohistochemical studies of human LBs have previously shown that LBs are stained with proteasome subunits (6) and molecular chaperones such as Hsp40 and Hsp70 (24). Thus, we next examined whether the inclusion bodies in COS-7 cells contain the 20 S proteasome core subunit and Hsp70. We found both the 20 S proteasome subunit and Hsp70 to be colocalized with synphilin-1 inclusion bodies (Fig. 2, G–L). Dorfin binds specifically to UbcH7 as an E2 through the RING finger/IBR domain (40). UbcH7 was also localized with Dorfin in these inclusions (Fig. 2, M–O). These observations suggest that large juxtanuclear inclusions formed by synphilin-1 in our cell culture system have many characteristic features of LBs, that synphilin-1 can aggregate when overexpressed, and that this process may be associated with its ubiquitylation.



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FIG. 2.
Formation of large juxtanuclear inclusions by overexpression of synphilin-1. Full-length synphilin-1 was overexpressed in COS-7 cells as DsRed fusion protein. Two days after transfection, cells were fixed and immunostained with the indicated antibodies. Large juxtanuclear inclusions of synphilin-1 were formed spontaneously without proteasome inhibition. Cells with large juxtanuclear inclusions were co-stained with ubiquitin (Ub) (A–C), Dorfin (D–F), 20 S proteasome core subunit (G–I), Hsp70 (J–L), or UbcH7 (M–O). Regions of overlap between synphilin-1 (red) and immunoreactivities of the indicated proteins (green) are shown in yellow. Nuclei were stained with Hoechst 33342 (blue). Scale bar = 10 µm.

 

Expression of the Central Portion of Synphilin-1 Induces Large Juxtanuclear Inclusions as Full-length Proteins, but Small Punctate Aggregates Are Also Formed—To further analyze which part of synphilin-1 is related to aggregation formation, we prepared a series of deletion mutants of synphilin-1. We divided synphilin-1 into three parts, the N terminus of synphilin-1 (synphilin-1-N) containing amino acids 1–348, the central portion of synphilin-1 (synphilin-1-M) containing amino acids 349–555, and the C terminus of synphilin-1 (synphilin-1-C) containing amino acids 556–919, and fused them to DsRed at their C termini (Fig. 3, A and B). Inclusions were not seen with overexpression of DsRed alone, synphilin-1-N, or synphilin-1-C in COS-7 cells (Fig. 3, C–E). However, expression of synphilin-1-M resulted in the production of two types of inclusions: large juxtanuclear inclusions (Fig. 3, F–K) and small punctate aggregates scattered throughout the cytoplasm (L–Q). The large inclusions were stained with ubiquitin (Fig. 3, F–H) and Dorfin (I–K), as were inclusions induced by full-length synphilin-1. However, neither ubiquitin nor Dorfin was colocalized with the small punctate aggregates scattered throughout the cytoplasm (Fig. 3, L–Q).



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FIG. 3.
Formation of two types of aggregates by the central portion of synphilin-1. COS-7 cells were transfected with expression vectors for DsRed alone or DsRed fusion proteins of synphilin-1 deletion mutants. Two days after transfection, cells were analyzed by Western blotting (WB) and immunocytochemistry. Shown are a schematic representation of the DsRed fusion proteins of synphilin-1 deletion mutants used in this study (A) and the results from Western blot analysis of lysates from transfected cells (B). DsRed alone (C), synphilin-1-N (D), and synphilin-1-C (E) formed no aggregates, whereas overexpression of the central portion of synphilin-1 (synphilin-1-M) induced two types of inclusions: large juxtanuclear inclusions (F and I) and small punctate aggregates scattered throughout the cytoplasm (L and O). Large juxtanuclear inclusions were ubiquitin (Ub)-positive (F–H) and colocalized with Dorfin (I–K), whereas small punctate aggregates were ubiquitin-negative (L–N) and did not colocalize with Dorfin (O–Q). Regions of overlap between synphilin-1 (red) and immunoreactivities of the indicated proteins (green) are shown in yellow. Nuclei were stained with Hoechst 33342 (blue). Scale bar = 10 µm.

 

Expression of the Central Portion of Synphilin-1 Compromises Cell Viability—We examined the frequency of the inclusion formation by synphilin-1 and its deletion mutants. The number of inclusions was counted with and without the proteasome inhibitor MG132 in COS-7 cells (Fig. 4A). Both synphilin-1-N and synphilin-1-C formed almost no inclusions in either the presence or absence of MG132. Full-length synphilin-1 and synphilin-1-M produced inclusions with high frequency even in the absence of MG132, and the number of cells with inclusions induced by full-length synphilin-1 was significantly greater than that induced by synphilin-1-M (Fig. 4A). Treatment with MG132 significantly increased the number of inclusions. We next measured the ratio of cells that contained small punctate aggregates to total cells bearing all inclusions (Fig. 4B) because two types of aggregates, large juxtanuclear inclusions and small punctate scattered aggregates, were observed. In contrast to full-length synphilin-1, the inclusions induced by overexpression of synphilin-1-M were predominantly small punctate aggregates scattered through the cytoplasm (Fig. 4B). Treatment with MG132 decreased the ratio of small aggregates induced by synphilin-1-M (Fig. 4B).



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FIG. 4.
The central portion of synphilin-1 produces predominantly small punctate aggregates and compromises cell viability. A, the frequency of inclusion-bearing cells transfected with synphilin-1 and its deletion mutants. COS-7 cells were grown on collagen-coated 4-chamber well slides and transfected with expression vectors for synphilin-1-DsRed fusion proteins. Two days after transfection, cells were fixed, and percentages of inclusion-positive cells among DsRed-positive cells were determined. For proteasome inhibition, cells were treated with 0.5 µM MG132 for 16 h before fixation. B, the frequency of cells bearing small punctate aggregates scattered through the cytoplasm among all inclusion-positive cells. Experimental conditions were same as described for A. Data are the means ± S.D. of triplicate assays. Statistical analyses were carried out with Mann-Whitney's U test. *, p < 0.01. C, the cytotoxic effect of synphilin-1-M expression in an MTS assay. Neuro2a cells were grown on collagen-coated 96-well plates and transfected with V5-tagged synphilin-1 or its deletion mutants. After changing to a serum-free medium, MTS assays were performed after 24 h of incubation. Viability of cells was measured as the level of absorbance at 490 nm. Data are means ± S.D. of triplicate assays. Statistical analyses were carried out by one-way analysis of variance. *, p < 0.01.

 

The effects of synphilin-1 expression on cell viability are poorly understood. O'Farrell et al. (36) reported that cells transfected with synphilin-1 are more viable than cells transfected with LacZ. On the other hand, Lee et al. (37) reported that synphilin-1 compromises cell viability. Thus, we examined the effects of synphilin-1 and its deletion mutants on cell viability using the MTS assay in the neuronal cell line Neuro2a (Fig. 4C). We found that synphilin-1-M had a cytotoxic effect, whereas overexpression of full-length synphilin-1 or the N- or C-terminal deletion mutant of synphilin-1 did not (Fig. 4C). We used synphilin-1-V5 fusion constructs, but synphilin-1-DsRed fusion constructs gave the same results (data not shown).

Dorfin Interacts with Synphilin-1—We examined whether Dorfin interacts with synphilin-1 because Dorfin localizes in LBs and cytoplasmic juxtanuclear inclusions formed by synphilin-1. To identify which portion of synphilin-1 binds to Dorfin, we expressed a series of deletion mutants of V5-tagged synphilin-1 and Xpress-tagged Dorfin in COS-7 cells (Fig. 5B). Co-immunoprecipitation confirmed that Dorfin bound to full-length synphilin-1 (Fig. 5A) and interacted strongly with synphilin-1-M and weakly with synphilin-1-N, but Dorfin failed to bind to synphilin-1-C (Fig. 5C). Thus, Dorfin interacts with synphilin-1 mainly through its central portion, which contains the ankyrin-like repeat, the coiled-coil domain, and the ATP/GTP-binding domain. Dorfin has a unique primary structure containing a RING finger/IBR motif at its N terminus and can be structurally divided into two parts, the N-terminal region containing a RING finger/IBR motif (Dorfin-N) that interacts with E2 and the C-terminal region with no similarity to any other known proteins (Dorfin-C) (Fig. 5B) (40). We found that Dorfin-C, but not Dorfin-N, specifically bound synphilin-1, indicating that Dorfin binds to synphilin-1 via its C-terminal region (Fig. 5D).



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FIG. 5.
Association of Dorfin with synphilin-1 in COS-7 cells. A, Dorfin binds to synphilin-1. V5-tagged synphilin-1 or LacZ was cotransfected with Xpress-tagged Dorfin in COS-7 cells. After immunoprecipitation (IP) was performed with anti-Xpress antibody, the resulting precipitates and cell lysate were analyzed by Western blotting (WB) with horseradish peroxidase-conjugated anti-V5 or anti-Xpress antibody. B, schematic representation of Xpress-tagged Dorfin, deletion mutants of Dorfin (i.e. Dorfin-N and Dorfin-C), V5-tagged synphilin-1, and deletion mutants of synphilin-1 (i.e. synphilin-1-N, synphilin-1-M, and synphilin-1-C) used in this study. C, Dorfin binds to synphilin-1 mainly through its central portion. After V5-tagged deletion mutants of synphilin-1 and Xpress-tagged Dorfin were transfected, immunoprecipitation and Western blotting were performed as described for A. D, binding of synphilin-1 to the C-terminal portion of Dorfin. After V5-tagged synphilin-1 and Xpress-tagged deletion mutants of Dorfin were transfected, immunoprecipitation and Western blotting were performed as described for A.

 

Dorfin Ubiquitylates Synphilin-1 through Its Central Domain In Vitro—The physical interaction between Dorfin and synphilin-1 prompted us to investigate whether synphilin-1 itself is ubiquitylated by Dorfin. We first examined whether synphilin-1 is ubiquitylated in a culture cell model. V5-tagged full-length synphilin-1 or its deletion mutants were cotransfected with FLAG-tagged ubiquitin in HEK293 cells. When full-length synphilin-1 or its deletion mutants were immunoprecipitated after treatment with the proteasome inhibitor MG132, full-length synphilin-1 and synphilin-1-M were found to be polyubiquitylated, but synphilin-1-N and synphilin-1-C were not (Fig. 6A). Wild-type and mutant {alpha}-synuclein were found not to be polyubiquitylated, whereas, as previously reported (44), mutant SOD1 was polyubiquitylated (Fig. 6A).



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FIG. 6.
Ubiquitylation of synphilin-1 by Dorfin. A, synphilin-1 is ubiquitylated in HEK293 cells. V5-tagged synphilin-1 or its deletion mutants were cotransfected with FLAG-tagged ubiquitin (Ub) in HEK293 cells and treated with 0.5 µM MG132 for 16 h overnight after transfection. Myc-tagged {alpha}-synuclein or SOD1 was cotransfected with FLAG-ubiquitin and treated as described above. Immunoprecipitates (IP) prepared with anti-V5 or anti-Myc antibody were used for immunoblotting with anti-FLAG antibody. B, in vitro ubiquitylation assay of synphilin-1 with Dorfin. Xpress-tagged Dorfin and V5-tagged synphilin-1 were transfected into HEK293 cells independently. Immunopurified Dorfin (IP-Xpress-Dorfin) and synphilin-1 (IP-synphilin-1-V5) were prepared and mixed in an assay mixture for ubiquitylation. For this assay, Myc-tagged wild-type (WT) and mutant {alpha}-synuclein and Myc-tagged mutant SOD1(G85R) were also used instead of synphilin-1. After a 90-min incubation at 30 °C, SDS-PAGE was performed, followed by Western blotting (WB) for His-tagged ubiquitin with anti-His antibody. C, in vitro ubiquitylation assay of various synphilin-1 deletion mutants with Dorfin. V5-tagged deletion mutants of synphilin-1 were transfected into HEK293 cells, immunopurified, and mixed with IP-Xpress-Dorfin in an assay mixture for ubiquitylation as described for B. The reaction products were analyzed by Western blotting with anti-His antibody for ubiquitin (left panel) and with anti-V5 antibody for synphilin-1 (right panel). High molecular mass ubiquitylated synphilin-1 and synphilin-1-M are shown as (Ub)n. Asterisks indicate IgG light and heavy chains.

 

We next examined whether Dorfin is involved in the ubiquitylation of synphilin-1 in vitro. For this purpose, we immunopurified Xpress-Dorfin and synphilin-1-V5 independently after transfection into HEK293 cells. When these immunopurified proteins were incubated with recombinant E1, E2 (UbcH7), His-tagged ubiquitin, and ATP, high molecular mass ubiquitylated bands were observed in the presence of Xpress-Dorfin with synphilin-1, whereas no signal was noted with synphilin-1 in the absence of either E1 or E2 (Fig. 6B). Dorfin ubiquitylated mutant SOD1 in vitro, as previously reported (44). Dorfin did not ubiquitylate either wild-type or mutant {alpha}-synuclein (Fig. 6B). In vitro ubiquitylation assay of a series of synphilin-1 deletion mutants with Dorfin revealed that synphilin-1-M was ubiquitylated, whereas synphilin-1-N and synphilin-1-C were not ubiquitylated at all (Fig. 6C).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Several lines of evidence have suggested that derangements in the ubiquitin-proteasome protein degradation pathway may have a prominent role in the pathogenesis of PD (5). Our present study shows that Dorfin, an E3 ubiquityl ligase, is colocalized with ubiquitin in LBs of PD and physically binds to ubiquitylate synphilin-1, which is known to be a major component of LBs (31, 38, 39).

For the analysis of LB formation by synphilin-1, various cell culture models have been reported (31, 3537). In our cell culture model, overexpression of synphilin-1 alone induced large juxtanuclear cytoplasmic inclusions. In these large inclusions, Dorfin was colocalized with ubiquitin/proteasome pathway-related proteins such as ubiquitin, the 20 S proteasome core subunit, and Hsp70, just as Dorfin in LBs. The central portion of synphilin-1 contains the ankyrin-like repeat, the coiled-coil domain, and the ATP/GTP-binding domain (31). This region is reported to be necessary for interaction with {alpha}-synuclein (32). We found that the central portion of synphilin-1 also bound with Dorfin and that overexpression of this region alone led to inclusion body formation, whereas neither the N- nor C-terminal regions induced aggregates. Overexpression of this central portion of synphilin-1 produced small punctate aggregates scattered throughout the cytoplasm as well as large juxtanuclear inclusions, but the former predominated. The small punctate aggregates did not colocalize with either ubiquitin or other proteasome pathway-associated proteins and had cytotoxic effects as revealed by MTS assays. Recently, Lee and Lee (50) reported that overexpression of {alpha}-synuclein in culture cells produces two distinct types of aggregates: large juxtanuclear inclusions and small punctate aggregates scattered throughout the cytoplasm. The juxtanuclear inclusion bodies are filled with amyloid-like {alpha}-synuclein fibrils, whereas the small aggregates contain non-fibrillar spherical aggregates (50). They suggested that these aggregates appear sequentially, with the smallest population appearing first and the fibrillar inclusions last, and that the small spherical aggregates are the cellular equivalents of protofibrils (50). Protofibrils are recognized to be more important in terms of cytotoxicity than mature fibrils in A{beta} (51, 52) and {alpha}-synuclein (53, 54). In our cell culture model, overexpression of synphilin-1 produced two distinct types of aggregates, very closely resembling two types of {alpha}-synuclein aggregates (50). Thus, the small punctate aggregates scattered throughout the cytoplasm induced by the central portion of synphilin-1 might have characteristics similar to those of protofibrils. Our cell culture system will allow detailed characterization of LB formation and cytotoxic processes in further studies.

We reported previously that Dorfin localizes in the inclusion bodies of familial ALS with SOD1 mutations as well as in those of sporadic ALS and ubiquitylates various SOD1 mutants derived from familial ALS patients (44). Based on these findings, it is conceivable that familial and sporadic forms of ALS share a common mechanism involving the dysfunction of the ubiquitin/proteasome pathway, despite having distinct etiological mechanisms. In sporadic ALS, unknown substrate(s) of Dorfin might play a role in the pathogenesis of the disease and accumulate in ubiquitylated inclusion bodies. The following results support the view that Dorfin plays an important role in the formation of LBs of PD: (i) the presence of Dorfin in LBs and large juxtanuclear inclusions of synphilin-1 in our cell culture model, (ii) the parallel distribution patterns of ubiquitin and Dorfin in LBs and inclusion bodies induced by synphilin-1 in cultured cells, and (iii) the E3 function of Dorfin to ubiquitylate synphilin-1. Dorfin did not ubiquitylate either wild-type or mutant {alpha}-synuclein; however, our results cannot exclude the possibility that post-translational modification, such as glycosylation (55) or phosphorylation (56, 57), of {alpha}-synuclein may be necessary for it to become a substrate for Dorfin because overexpressed {alpha}-synuclein was not phosphorylated in our cell culture system (data not shown). The relation between Dorfin and PD shows striking similarities to the relation between Dorfin and ALS. Our findings raise the possibility that PD and ALS are etiologically distinct, but share a biochemically common metabolic pathway through Dorfin, leading to the formation of ubiquitylated inclusion bodies and to neuronal cell degeneration.

Parkin has been shown to have E3 ubiquityl ligase activity (1012). It was recently demonstrated that an O-glycosylated {alpha}-synuclein (55) and synphilin-1 (35) are the substrates of parkin and that parkin localizes to LBs of sporadic PD (49). The link between sporadic and familial forms of PD through {alpha}-synuclein, synphilin-1, and parkin sheds new light on underlying common molecular pathogenic mechanisms in PD. What roles, then, do Dorfin and parkin play with respect to each other in the pathogenesis of PD and/or LB formation? Both proteins have a RING finger/IBR domain and E3 ubiquitin ligase activities. Parkin interacts with both {alpha}-synuclein (55) and synphilin-1 (35), whereas Dorfin binds and ubiquitylates only synphilin-1. Parkin resides in the core of LBs (49), whereas Dorfin localizes predominantly to the rim. Impaired function of parkin as an E3 ubiquityl ligase is responsible for one of the most common forms of familial PD, autosomal recessive juvenile parkinsonism (9, 10). However, there has been no analysis of whether Dorfin gene mutation causes familial PD. Recently, Valente et al. (58, 59) identified a locus, PARK6, on chromosome 1p35 [PDB] –1p36 that is involved in the autosomal recessive form of parkinsonism. Interestingly, a human paralog of Dorfin (Dj1174N9.1) has been mapped at 1p34.1–1p35.3 (60). Furthermore, Dorfin was identified by a phage display system to be one of the binding proteins with 2-methylnorharman, an analog of the parkinsonism-inducing toxin, 1-methyl-4-phenylpyridinium cation (61). These findings suggest the utility of analyzing Dj1174N9.1 or Dorfin mutation for potential involvement in familial PD. Production of Dorfin knockout mice will also answer the question of whether Dorfin is essential for pathogenesis and/or ubiquitylated inclusion body formation in PD.


    FOOTNOTES
 
* This work was supported in part by a Center of Excellence grant from the Ministry of Education, Culture, Sports, Science, and Technology and by grants from the Ministry of Health, Labor, and Welfare of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Research Fellow of the Japan Society for the Promotion of Science for Young Scientists. Back

§ To whom correspondence should be addressed: Dept. of Neurology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan. Tel.: 81-52-744-2385; Fax: 81-52-744-2384; E-mail: sobueg{at}med.nagoya-u.ac.jp.

1 The abbreviations used are: PD, Parkinson's disease; LBs, Lewy bodies; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; HEK293, human embryonic kidney 293; ALS, amyotrophic lateral sclerosis; SOD1, superoxide dismutase-1; IP, immunopurified; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt. Back


    ACKNOWLEDGMENTS
 
We thank Dr. Miya Kobayashi (Kinjo Gakuin University) for helpful comments.



    REFERENCES
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 ABSTRACT
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