Human Molecular Genetics

This Article Abstract FREE Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Search for citing articles in: ISI Web of Science (66) Request Permissions Google Scholar Articles by Marx, F. P. Articles by Krüger, R. Search for Related Content PubMed PubMed Citation Articles by Marx, F. P. Articles by Krüger, R. Social Bookmarking          What's this?

Human Molecular Genetics, 2003, Vol. 12, No. 11 1223-1231
DOI: 10.1093/hmg/ddg134
© 2003 Oxford University Press

Identification and functional characterization of a novel R621C mutation in the synphilin-1 gene in Parkinson's disease

Frank P. Marx¹, Carsten Holzmann², Karsten M. Strauss¹, Lei Li^2,3, Olaf Eberhardt¹, Ellen Gerhardt¹, Mark R. Cookson⁴, Dena Hernandez⁴, Matt J. Farrer⁵, Jennifer Kachergus⁵, Simone Engelender⁶, Christopher A. Ross⁷, Klaus Berger⁸, Ludger Schöls⁹, Jörg B. Schulz¹, Olaf Riess¹⁰ and Rejko Krüger^1,*

¹Department of Neurology, Laboratory of Neurodegeneration, University of Tübingen, Tübingen, Germany, ²Department of Medical Genetics, University of Rostock, Rostock, Germany, ³Department of Traditional Chinese Medicine, Union Hospital, Tongji Medical University, Wuhan, People's Republic of China, ⁴Laboratory of Neurogenetics, National Institute on Aging, Bethesda, MD, USA, ⁵Neurogenetics Laboratory, Mayo Clinic, Jacksonville, USA, ⁶Department of Pharmacology, B. Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel, ⁷Department of Psychiatry, Division of Neurobiology, Johns Hopkins University School of Medicine, Baltimore, MD, USA, ⁸Department of Epidemiology and Social Medicine, University of Münster, Germany, ⁹Neurology, University of Bochum, Bochum, Germany and ¹⁰Department of Medical Genetics, University of Tübingen, Tübingen, Germany

Received February 4, 2003; Accepted March 21, 2003

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Synphilin-1 is linked to the pathogenesis of Parkinson's disease (PD) based on its identification as an -synuclein (PARK1) and parkin (PARK2) interacting protein. Moreover, synphilin-1 is a component of Lewy bodies (LB) in brains of sporadic PD patients. Therefore, we performed a detailed mutation analysis of the synphilin-1 gene in 328 German familial and sporadic PD patients. In two apparently sporadic PD patients we deciphered a novel C to T transition in position 1861 of the coding sequence leading to an amino acid substitution from arginine to cysteine in position 621 (R621C). This mutation was absent in a total of 702 chromosomes of healthy German controls. To define a possible role of mutant synphilin-1 in the pathogenesis of PD we performed functional analyses in SH-SY5Y cells. We found synphilin-1 capable of producing cytoplasmic inclusions in transfected cells. Moreover we observed a significantly reduced number of inclusions in cells expressing C621 synphilin-1 compared with cells expressing wild-type (wt) synphilin-1, when subjected to proteasomal inhibition. C621 synphilin-1 transfected cells were more susceptible to staurosporine-induced cell death than cells expressing wt synphilin-1. Our findings argue in favour of a causative role of the R621C mutation in the synphilin-1 gene in PD and suggest that the formation of intracellular inclusions may be beneficial to cells and that a mutation in synphilin-1 that reduces this ability may sensitize neurons to cellular stress.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Genetic studies identified mutations in -synuclein and ubiquitin C-terminal hydrolase as rare causes of autosomal-dominant Parkinson's disease (PD) and mutations in parkin as a cause of autosomal recessive PD (1–4). Subsequently, the respective proteins were identified as major components of Lewy bodies, proteinaceous intracytoplasmic inclusions in affected brain regions, in hereditary but also in the more common sporadic form of PD. Functional characterization of the identified genes implicated the ubiquitin-mediated protein degradation pathway in the pathogenesis of PD (5–7). The identification of synphilin-1 as both an -synuclein-interacting protein and a component of LB in brains of sporadic PD patients provided an important functional link between synphilin-1 and neurodegeneration in PD (8,9). Synphilin-1 is a 919 amino acid protein of which the function is currently unknown. It is predominantly expressed in neurons, localized in the cytosol and presynaptic nerve terminals and associates with synaptic vesicles. Thus, synphilin-1 shares the same intracellular compartments as -synuclein and parkin (10). Synphilin-1 contains several motifs propagating protein–protein interactions, namely ankyrin-like repeats and a coiled-coil domain. A potential role in pathological protein aggregation has been supported by co-transfection experiments of synphilin-1 with NAC, a fragment of -synuclein, leading to eosinophilic intracytoplasmic aggregates in HEK293 cells (8).

Based on a whole genome mapping approach in large cohorts of PD patients in three independent studies, a candidate locus for PD was identified on the long arm of chromosome 5 containing the synphilin-1 gene (11–13). This made synphilin-1 a candidate for mutation screening in sporadic and familial PD patients and led us to characterize the functional role of synphilin-1 in dopaminergic neuronal cell lines concerning its influence on the formation of protein aggregates and cell viability.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Mutation screening
Direct sequencing revealed one intronic (A to G at position -69, 5' of the first translated exon) and five exonic base pair substitutions: (i) C636T in exon 3 (ctttctccYgtgaaaag); (ii) C1134T in exon 4 (gactgcctYaatgagcg); (iii) C1510T in exon 7 (ggcacaccYtgtgctcc); (iv) C1861T in exon 9 (aaatcttaYgccagtta); and (v) G2125C in exon 9 (aaagcgtaSagagtatg). Whereas the first three exonic substitutions are silent polymorphisms, the C1861T substitution leads to an amino acid exchange from arginine to cysteine in position 621 of the peptide sequence (R621C) and the G2125C substitution to an amino acid exchange from glutamate to glutamine in position 709 of the coding sequence (E709Q). The G2125C transversion creates a new Csp6I restriction site. Performing an association study in our PD patients and 351 healthy controls we found no significant differences in allelic or genotypic distribution between the two groups (Table 1). In two apparently sporadic PD patients we identified the transition from C to T in position 1861 of the coding sequence leading to an amino acid substitution from arginine to cysteine in position 621 of the peptide sequence (R621C; Fig. 1). This sequence variation was absent in a total of 702 chromosomes of healthy German controls. Both patients were included in our study as sporadic because there was no family history for PD. However genotyping of six genetic markers in the chromosomal region harbouring the synphilin-1 gene revealed that, concerning five microsatellite markers, both patients share the same rare alleles (Table 2).

View this table: [in this window] [in a new window]   Table 1. Identified coding sequence alterations leading to changes in the peptide sequence of synphilin-1. Frequencies of the respective variants in PD patients and control individuals

 

View larger version (39K): [in this window] [in a new window]   Figure 1. Identification of the R621C mutation in the synphilin-1 gene. The band shift on the SSCP gel (arrow) was identified as a heterozygous base exchange from C to T in position 1861 of the coding sequence by direct sequencing.

 

View this table: [in this window] [in a new window]   Table 2. Genotyping of genetic markers spanning the synphilin-1 locus in carriers of the R621C mutation and controls

 
Patient I
A 66-year-old man first presented with a 3 year history of progressive slowing of movement and painful legs. Neurological examination revealed an asymmetric akinetic rigid Parkinson syndrome with a masked face, reduced arm swinging while walking, mild anteflexion, slow short-stepped gait and rigidity pronounced on the left side. No tremor, oculomotor dysfunction or cognitive deficits developed. Reduced urinary flow, some urinary urgency and nycturia twice a night were assigned to benign prostatic hypertrophy. Arterial hypotension had been recognized since adolescence but did not decompensate with dopaminergic therapy. L-dopa therapy resulted in substantial and long lasting benefit. Even after 11 years of dopaminergic treatment fluctuations, dyskinesias or psychosis did not develop. He still managed all activities of daily living including snow shovelling at the age of 74 years. Family history (six sibs and three children) was negative for movement disorders with the parents dying at 77 and 82 years of age, respectively.

Patient II
The second patient, a 71-year-old man, recognized difficulties in movements of his left arm at 69 years of age. Two years later rest tremor of the left hand developed and he complained of muscular pain in the left shoulder. Cogwheel rigidity and impairment of fine motor skills were pronounced on the left. Gait was slowed with anteflexion and reduced arm swinging. Apart from congenital nystagmus no oculomotor abnormality was observed. After prostatic surgery at the age of 69 years bladder function was normal. Neuropsychological function was unremarkable apart from an obsessive–compulsive personality structure. Response to L-dopa was pronounced and without complications for years. Family history disclosed no movement disorders. Since this patient deceased in the meantime no further information on the family was available and re-evaluation of the pedigree for a common ancestor with the other mutation carrier could not be performed.

Expression of synphilin-1-EGFP fusion proteins in HEK293 and SH-SY5Y cells
HEK293 cells transfected with cDNA encoding wild-type (wt; R621C) or mutant (C621) synphilin-1-EGFP fusion protein produced a band of 130 kDa on western blots when immunoblotted using anti-synphilin-1 polyclonal antibodies (Fig. 2). This is the predicted size based on the amino acid sequences of synphilin-1 (GenBank accession number NP_005451; 100 kDa) and EGFP (27 kDa) and was confirmed using a monoclonal antibody to GFP (data not shown). Transient transfection of SH-SY5Y cells with EGFP-tagged synphilin-1 resulted in the formation of one or more cytoplasmic inclusions in a subset of cells (Fig. 3A and B), whereas controls expressing EGFP alone displayed a homogeneous signal without any inclusions (Fig. 3C). Similar results were obtained in HEK293 cells (data not shown). Comparing the R621 synphilin-1 and C621 synphilin-1 variants in otherwise-untreated SH-SY5Y cells, we found a 0.78-fold decreased inclusion formation in cells transfected with the C621 synphilin-1-EGFP construct (Fig. 4).

View larger version (16K): [in this window] [in a new window]   Figure 2. Expression of wt and mutant synphilin-1. HEK293 cells transfected with cDNA encoding wt (R621) or mutant (C621) synphilin-1-EGFP fusion protein produced a band of expected size on western blots (lanes 1 and 2). Untransfected HEK293 cells served as controls (lane 3). Equal amounts of protein were resolved on SDS–PAGE gel and subjected to western blot analysis using anti-synphilin-1 polyclonal antibodies (upper panel) and anti-actin polyclonal antibodies as loading control (lower panel).

 

View larger version (37K): [in this window] [in a new window]   Figure 3. Formation of intracytoplasmic synphilin-1 inclusions in SH-SY5Y cells. SH-SY5Y cells were transfected for 48 h with control vector (pEGFPN1), wt synphilin-1 (pEGFPN1-R621 synphilin-1) or mutated synphilin-1 (pEGFPN1-C621 synphilin-1). A subset of cells overexpressing R621 or C621 synphilin-1 displayed one or more intracytoplasmic synphilin-1 inclusions (A and B), whereas cells expressing EGFP alone showed a homogenous distribution of the protein in the cytoplasm (C).

 

View larger version (15K): [in this window] [in a new window]   Figure 4. Proportion of cells bearing intracytoplasmic synphilin-1 inclusions. Overexpressing R621 synphilin-1 and C621 synphilin-1 variants in otherwise untreated SH-SY5Y cells, we found a decreased inclusion formation in cells transfected with the C621 synphilin-1-EGFP construct. After treatment with lactacystin the number of inclusions increased in cells overexpressing wt or C621 synphilin-1. The number of inclusion-bearing cells differed significantly between wt synphilin-1 or mutated synphilin-1 expressing cells. There was a significantly lower number of inclusion bearing cells in C621 synphilin-1 expressing SH-SY5Y cells at P<0.00015 by Student's-t-test. Results are indicated as mean±SD of three unrelated cultures, each experiment was replicated twice with similar results.

 
Effects of proteasome inhibition on inclusion formation
In order to test the effect of proteasome inhibition on inclusion formation, we treated transfected SH-SY5Y cells with 4 µM clasto-lactacystin ß-lactone (Calbiochem, Germany) for 4 h. Treatment with lactacystin resulted in an increase in the number of cells containing cytoplasmic synphilin-1 inclusions, which was more pronounced for wt synphilin-1 (Fig. 4). The proportion of inclusion bearing cells was significantly lower in cells expressing mutant (C621) synphilin-1 compared to wt (R621) synphilin-1 (Student's-t-test, P<0.00015; Fig. 4).

Effects of synphilin-1 expression on cell viability
To test the effect of synphilin-1 on cell viability we determined the proportion of apoptotic and necrotic cells in untreated and staurosporine-treated transfected SH-SY5Y cells by FACS analysis. Untreated R621 and C621 synphilin-1 transfected cells displayed a similar fraction of 90% viable cells. After treatment with 0.5 µM staurosporine for 6 h, cells expressing C621 synphilin-1 were more susceptible to the apoptotic stimulus with a decreased cell viability of 61% compared with 71% in cells expressing wt synphilin-1 (Fig. 5). This decreased viability of cells expressing mutant synphilin-1 was mainly due to an increase in the proportion of apoptotic cells from 4.5 to 14% (Fig. 5).

View larger version (55K): [in this window] [in a new window]   Figure 5. Viability of wt and C621 synphilin-1 transfected cells. FACS analysis of SH-SY5Y cells transiently transfected with wt or C621 synphilin-1 using propidium iodide (PI) and annexin-Cy5 as markers of necrosis and apoptosis, respectively. Cells positive for propidium iodide staining are displayed in the upper left (UL) quadrant, whereas annexin-Cy5-positive cells are displayed in the lower right (LR) quadrant. Cells at later stages of cell death, which show alterations typical for both apoptosis and necrosis, are displayed in the upper-right area. Untreated cells expressing either R621 synphilin-1 or C621 synphilin-1 show similar distributions of the respective cell fractions 48 h after transfection (A and C). After treatment with 0.5 µM staurosporine for 6 h cells expressing C621 synphilin-1 showed decreased viability to 61% compared with 71% in cells expressing wt-synphilin-1 (B and D).

 
Interaction of wt and mutant synphilin-1 with -synuclein and parkin
To delineate a possible effect of the R621C mutation on the interaction of synphilin-1 with known interacting proteins, we performed co-transfection experiments of wt and C621 synphilin-1 with -synuclein and parkin in HEK293 cells. This was followed by co-immunoprecipitation using FLAG-tagged synphilin-1 and myc-tagged -synuclein and parkin constructs. We confirmed the reported interaction of synphilin-1 with parkin and -synuclein. No difference in the ability to interact was observed between wt and C621 synphilin-1 (Fig. 6). The same result was obtained with A30P -synuclein (data not shown). To further delineate a possible effect of mutant synphilin-1 on the interaction with -synuclein, we tested the effect of wild-type and C621 synphilin-1 on inclusion formation 48 h after co-transfection with -synuclein. We found no consistent effect on the proportion of inclusion-bearing cells comparing co-transfection of either wild-type or C621 synphilin-1 (data not shown).

View larger version (43K): [in this window] [in a new window]   Figure 6. Interaction of wt and C621 synphilin-1 with -synuclein and parkin. Lysates prepared from HEK293 cells transfected with FLAG-tagged synphilin-1 (wt or C621) and myc-tagged parkin or -synuclein, respectively, were subjected to IP with anti-FLAG and subsequently immunoblotted with anti-myc antibodies. The blots were also probed with anti-FLAG antibodies to illustrate relatively equal amounts of synphilin-1 expression. Wild-type synphilin-1 and C621 synphilin-1 specifically co-precipitated -synuclein (A) and parkin (B) when co-transfected in HEK293 cells. No evidence for differential interaction of the respective proteins could be observed.

 

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Neurodegeneration in PD is closely linked to protein aggregation and disturbed protein degradation. Synphilin-1 has been identified as an -synuclein interacting protein and a recent study defined synphilin-1 as a substrate for the ubiquitin E3 ligase parkin (14). Thus synphilin-1 provides a functional link between -synuclein and parkin, both involved in PD, making it an excellent candidate for mutation screening in PD. Two earlier studies sequencing the synphilin-1 gene in index patients from a total of 69 PD families found no coding changes and failed to provide genetic evidence for a role of synphilin-1 in PD (15,16). Here we report the first coding substitution, a novel R621C mutation in the synphilin-1 gene in two apparently independent patients with sporadic PD. As indicated by recent studies on genetic susceptibility factors in the common late-onset form of PD, the sporadic appearance of the disease does not exclude an involvement of genetic factors in the pathogenesis of the disease (13). Extensive genealogical effort in the Icelandic population showed that, although most PD patients do not exhibit affected first-degree relatives, for many of them affected relatives could be identified (13). These affected relatives were even found at six meiotic events illustrating difficulties in defining a genetic contribution to PD based on the limited pedigree size in central European families. Evidence for a possible relationship between both R621C mutation carriers came from genotyping of genetic markers in and around the synphilin-1 gene. Our results of shared rare alleles in five out of six tested microsatellite markers argue in favour of a common founder. Therefore, current genealogical investigations centre on the identification of a possible common ancestor of the two index patients. Support for a possible role of the R621C mutation in the pathogenesis of PD came firstly from interspecies comparison of the amino acid sequence of the synphilin-1 protein. Synphilin-1 harbours a coiled-coil and an ankyrin-like domain, which are known to be involved in protein–protein interactions and which are highly conserved among mouse and humans. Moreover there is a large homologous stretch in both species from amino acid position 612–689 of unknown function (Fig. 7). The presence of the arginine to cysteine substitution in a region that is identical between different species suggests functional implications (17).

View larger version (12K): [in this window] [in a new window]   Figure 7. Schematic view of the synphilin-1 protein. Schematic view of the synphilin-1 protein including an interspecies comparison of the amino acid (aa) sequence of synphilin-1, which indicates the localization of the R621C mutation in a highly conserved region among mouse and human.

 
We found an aggregation-forming capacity of both wt and mutant synphilin-1. Our results of an increase in synphilin-1 aggregation after treating SH-SY5Y cells with the proteasome inhibitor lactacystin suggest that synphilin-1 is physiologically degraded by the proteasome. We have shown previously that another proteasome inhibitor, MG132, increases the tendency of synphilin-1 to form inclusions in HEK293 cells transiently transfected with synphilin-1 (18), although in other stable cell lines, inclusions are not seen until after treatment with proteasome inhibitors (19). There is an increase of high-molecular-weight ubiquitinated synphilin-1 after treatment with proteasome inhibitors (14), a slowing of synphilin-1 turnover and an increase in steady-state protein levels (19). The fact that wt synphilin-1 transfected cells are more sensitive to proteasome inhibition than C621 synphilin-1 transfected cells might indicate differences in the recognition by the ubiquitin–proteasome system.

The decreased capacity of mutant synphilin-1 to aggregate is accompanied by an increased susceptibility to apoptotic stimuli in viability assays, indicating a novel function of the mutant C621 synphilin-1. Taking the typical late onset of PD in carriers of the R621C mutation into account, this argues in favour of a mild, long-lasting toxic gain of function leading to neurodegeneration. Thus, based on its apparently sporadic appearance, the R621C substitution has to be regarded as a genetic susceptibility factor or genetic trait with reduced penetrance. In our study we found no evidence that the toxic gain of function of the C621 synphilin-1 may be mediated by differential interaction with the known synphilin-1 interacting proteins, -synuclein and parkin. Indeed the domain containing the R621C mutation seems not to be involved in the interaction with -synuclein and parkin: studies defining the critical interacting domains of synphilin-1 did not reveal significant interaction with -synuclein and parkin using fragments harbouring amino acid residues 466–711 and 556–919, respectively (14,20).

Our results support the hypothesis of Lansbury and colleagues (21) dissociating the formation of protein aggregates from cell death and suggest that in fact protein aggregates or intracellular inclusions can be beneficial for cell survival. This is supported by several lines of evidence: (i) overexpression of -synuclein in animal models leads to dopaminergic cell death and motor dysfunction without formation of fibrillar protein aggregates (22); (ii) the majority of PD patients carrying parkin mutations display no proteinaceous inclusions in affected brain regions, however neuronal loss and clinical symptoms appear earlier in life than in idiopathic PD (23); and (iii) studies in brains of PD patients show that the majority of neurons in the substantia nigra undergoing apoptotic cell death do not contain LB, indicating that LB do not predispose to apoptotic-like cell death (24). Indeed some of us found a possible beneficial effect of synphilin-1 aggregates in HEK293 cells, since on a cellular level inclusion-bearing cells rarely displayed signs of cell death (18). Thus we speculate that the C621 mutation in the synphilin-1 gene prevents its sequestration into intracytoplasmic aggregations and leads to an increased accumulation of toxic intermediates by defective ubiquitination and/or proteasomal inhibition. Future studies will centre on the definition and functional characterization of the non-aggregated fraction of synphilin-1.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Patients and controls
A total of 328 German PD patients (mean age 66.3 years, SD±10.8 years; mean age at disease onset 55.9 years, SD±11.45 years; males 57.8%, females 42.2%) were evaluated by neurologists and were diagnosed as idiopathic PD based on the UK PD brain bank criteria, except for the criterion of absent family history. Based on pedigree analysis 56 patients had a positive family history of PD; the other patients were thought to be sporadic. All subjects signed an informed consent. Controls were 351 healthy German individuals of the MEMO-Study (Memory and Morbidity in Augsburg Elderly) (25) without cardinal signs for PD based on standardized UPDRS protocols (26) (mean age 72±4.3 years; males 52%, females 48%). Cardinal signs of PD were defined as UPDRS score 2 for tremor, rigidity or hypokinesia and resulted in exclusion from the controls.

PCR conditions
PCR was carried out in a thermocycler (Robocycler, Stratagene; Perkin Elmer 9700, PE Biosystems, USA) under the following conditions: 50 ng DNA was amplified in a final volume of 10 µl in the presence of 10 mM Tris–HCl (pH 8.3), 50 mM KCl, 2 mM MgCl₂, 200 µM of each dNTP, 10 pmol of each PCR primer, 1 µCi [32P]dCTP and 1 U Taq polymerase (Genecraft, Germany). Primers used and cycling conditions are summarized in Table 3. Whenever the PCR product length exceeded 280 bp, this step was followed by incubation with the proper restriction enzyme in order to increase the sensitivity of the single strand conformation polymorphism analysis (SSCP) analysis (27).

View this table: [in this window] [in a new window]   Table 3. Mutation analysis of the coding region and adjacent intronic sequences of the synphilin-1 gene: PCR conditions and restriction fragments

 
SSCP analysis and sequencing
DNA samples were electrophoresed on non-denaturating polyacrylamide gels using two different conditions [5% acrylamide (AA), 0.5x TBE, 5% glycerol and 6% AA, 1x TBE, 10% glycerol]. SSCP gels were visualized using autoradiography. DNA samples exhibiting bandshifts on the SSCP gel were sequenced on an ABI 310 apparatus using the BigDye Cycle Sequencing Kit (PE Biosystems, USA).

Cell culture, transfection and western blotting
Human neuroblastoma SH-SY5Y cells and human embryonic kidney 293 (HEK293) cells were cultured in Dulbecco's modified eagle's medium (DMEM; Invitrogen/Gibco, Belgium) supplemented with 10% fetal bovine serum (FBS; Invitrogen/Gibco, Belgium) and penicillin and streptomycin 1%, respectively (PAN, Germany). Cells were grown at 37°C in a humidified atmosphere containing 5% CO₂.

The cloning of human synphilin-1 into the pEGFPN1 expression vector has been described previously (18). Mutagenesis to introduce the R621C amino acid change was performed using the transformer site directed mutagenesis kit (Clontech, USA) and plasmids were resequenced to verify their identity. FLAG-tagged expression constructs of R621 and C621 synphilin-1 were generated by PCR using primers introducing a 5' FLAG tag. The constructs were cloned into pAdTrack-CMV vector (28) between XhoI and HindIII sites. Full-length cDNA of parkin was cloned into pcDNA3.1-myc₆ vector between BamHI and XbaI sites to generate pcDNA3.1-myc₆-parkin. The pcDNA3.1-myc₆ vector was generated by inserting a 6x myc-tag between HindIII and BamHI sites of pcDNA3.1 vector (Invitrogen, USA). Full-length cDNAs of -synuclein and A30P -synuclein were cloned into pcDNA3.1-myc₆ vector between EcoRV and XbaI sites to generate pcDNA3.1-myc₆--synuclein or pcDNA3.1-myc₆-A30P -synuclein, respectively. The integrity of the constructs was confirmed by sequencing. Transient transfection was carried out in six-well plates using Effectene transfection protocol (QiaGen, Germany) for SH-SY5Y cells or FuGENE 6 transfection protocol (Roche, Germany) for HEK293 cells, respectively. For western blots, HEK293 cells were transiently transfected with 2 µg of the target vector. For fluorescence microscopy, SH-SY5Y or HEK293 cells were transiently transfected with 1 µg of the target vector.

Total cellular protein extracts of HEK293 cells were prepared 48 h after transfection. Cells were washed with cold PBS and harvested in lysis buffer (PBS, 1% Triton X-100 and Complete protease inhibitor; Roche, Germany). After incubation for 30 min at 4°C the lysate was centrifuged at 13 000 rpm (Heraeus no. 3325) for 30 min at 4°C to remove insoluble matters. Total protein concentrations were measured using Bio-Rad protein assay kit (Bio-Rad, Germany). Equal amounts of protein (30 µg per lane) were resolved on SDS–PAGE gel and subjected to western blot analysis using a rabbit polyclonal antibody raised against amino acids 83–99 of synphilin-1 (18) and a goat anti-actin polyclonal antibody (Santa Cruz Biotechnology, USA).

Co-immunoprecipitation
HEK293 cells were transiently transfected with 2 µg of each vector. Forty-eight hours after transfection cells were washed with cold PBS and harvested in immunoprecipitation buffer (PBS, 0.5% Triton X-100 and Complete protease inhibitor; Roche, Germany). After incubation for 30 min at 4°C the lysates were centrifuged at 13 000 rpm (Heraeus no. 3325) for 30 min at 4°C to remove insoluble matters. Forty microlitres of ANTI-FLAG M2 affinity gel (Sigma, Germany) were added to the supernatants followed by rotating overnight at 4°C. The ANTI-FLAG M2 affinity gel was pelleted and washed three times using immunoprecipitation buffer. The precipitates were resolved on SDS–PAGE gel and subjected to western blot analysis using a rabbit anti-FLAG polyclonal antibody (Sigma, Germany) and a mouse anti-myc monoclonal antibody (Santa Cruz Biotechnology, USA).

Fluorescence microscopy
Cells transfected with pEGFPN1, pEGFPN1-R621 synphilin-1 or pEGFPN1-C621 synphilin-1 were examined for EGFP fluorescence without fixation 48 h after transfection on an inverted fluorescence microscope (DM IRBE, Leica, Germany). Counts of cells containing inclusions were performed by an investigator blinded to the transfection. More than 300 cells in randomly selected microscopic fields were counted in each of three independent cultures. Each experiment was replicated twice.

Viability assays
Cells were transfected with pEGFP-R621 synphilin-1 or pEGFP-C621 synphilin-1. Forty-eight hours after transfection cells were analyzed by flow cytometry on a FACScalibur (BD Biosciences, USA) using propidium iodide (Sigma, Germany) and annexin-Cy5 (BD Pharmingen, USA) staining to determine the proportions of necrotic and apoptotic cells respectively. Cells were gated for EGFP positivity and subsequently analysed for propidium iodide and annexin-Cy5 fluorescence measured as emissions at 600 and 670 nm, respectively. We counted more than 7000 cells for each group, experiments were performed three times with similar results.

	ACKNOWLEDGEMENTS

 
We thank S. Kautzmann for excellent technical assistance and C. O'Farrell for providing us with polyclonal synphilin-1 antibodies. This work was supported by a grant from the Federal Ministry of Education and Research (Fö 01KS9602) and the Interdisciplinary Center of Clinical Research Tübingen (IZKF) to R.K., by a grant of the German Research Society (DFG; Scho754/21) to L.S. and R.K, and by a NINDS grant (NS 38377) to C.R. L.L. is supported by a fellowship of the German Academic Exchange Service (DAAD).

	FOOTNOTES

 
^* To whom correspondence should be addressed at: Department of Neurology, Laboratory of Neurodegeneration, University of Tübingen, Hoppe-Seyler-Str. 3, D-72076 Tübingen, Germany. Tel: +49 70712982141; Fax: +49 7071295260; Email: rejko.krueger{at}uni-tuebingen.de

	REFERENCES

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 

1. Polymeropoulos, M.H., Lavedan, C., Leroy, E., Ide, S.E., Dehejia, A., Dutra, A., Pike, B., Root, H., Rubenstein, J., Boyer, R. et al. (1997) Mutation in the alpha-synuclein gene identified in families with Parkinson's disease. Science, 276, 2045–2047.[Abstract/Free Full Text]

2. Kitada, T., Asakawa, S., Hattori, N., Matsumine, H., Yamamura, Y., Minoshima, S., Yokochi, M., Mizuno, Y. and Shimizu, N. (1998) Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism. Nature, 392, 605–608.[CrossRef][Medline]

3. Krüger, R., Kuhn, W., Muller, T., Woitalla, D., Graeber, M., Kosel, S., Przuntek, H., Epplen, J.T., Schols, L. and Riess, O. (1998) Ala30Pro mutation in the gene encoding alpha-synuclein in Parkinson's disease. Nat. Genet., 18, 106–108.[CrossRef][Web of Science][Medline]

4. Leroy, E., Boyer, R., Auburger, G., Leube, B., Ulm, G., Mezey, E., Harta, G., Brownstein, M.J., Jonnalagada, S., Chernova, T. et al. (1998) The ubiquitin pathway in Parkinson's disease. Nature, 395, 451–452.[CrossRef][Medline]

5. McNaught, K.S., Olanow, C.W., Halliwell, B., Isacson, O. and Jenner, P. (2001) Failure of the ubiquitin-proteasome system in Parkinson's disease. Nat. Rev. Neurosci., 2, 589–594.[CrossRef][Web of Science][Medline]

6. Krüger, R., Eberhardt, O., Riess, O. and Schulz, J.B. (2002) Parkinson's disease: one biochemical pathway to fit all genes? Trends Mol. Med., 8, 236–240.[CrossRef][Web of Science][Medline]

7. Petrucelli, L., O'Farrell, C., Lockhart, P.J., Baptista, M., Kehoe, K., Vink, L., Choi, P., Wolozin, B., Farrer, M., Hardy, J. et al. (2002) Parkin protects against the toxicity associated with mutant alpha-synuclein: proteasome dysfunction selectively affects catecholaminergic neurons. Neuron, 36, 1007–1019.[CrossRef][Web of Science][Medline]

8. Engelender, S., Kaminsky, Z., Guo, X., Sharp, A.H., Amaravi, R.K., Kleiderlein, J.J., Margolis, R.L., Troncoso, J.C., Lanahan, A.A., Worley, P.F. et al. (1999) Synphilin-1 associates with alpha-synuclein and promotes the formation of cytosolic inclusions. Nat. Genet., 22, 110–114.[CrossRef][Web of Science][Medline]

9. Wakabayashi, K., Engelender, S., Yoshimoto, M., Tsuji, S., Ross, C.A. and Takahashi, H. (2000) Synphilin-1 is present in Lewy bodies in Parkinson's disease. Ann. Neurol., 47, 521–523.[CrossRef][Web of Science][Medline]

10. Ribeiro, C.S., Carneiro, K., Ross, C.A., Menezes, J.R. and Engelender, S. (2002) Synphilin-1 is developmentally localized to synaptic terminals, and its association with synaptic vesicles is modulated by alpha-synuclein. J. Biol. Chem., 277, 23927–23933.[Abstract/Free Full Text]

11. Scott, W.K., Nance, M.A., Watts, R.L., Hubble, J.P., Koller, W.C., Lyons, K., Pahwa, R., Stern, M.B., Colcher, A., Hiner, B.C. et al. (2001) Complete genomic screen in Parkinson disease: evidence for multiple genes. JAMA, 286, 2239–2244.[Abstract/Free Full Text]

12. Pankratz, N., Nichols, W.C., Uniacke, S.K., Halter, C., Rudolph, A., Shults, C., Conneally, P.M. and Foroud, T. (2002) Genome screen to identify susceptibility genes for Parkinson disease in a sample without parkin mutations. Am. J. Hum. Genet., 71, 124–135.[CrossRef][Web of Science][Medline]

13. Hicks, A.A., Petursson, H., Jonsson, T., Stefansson, H., Johannsdottir, H.S., Sainz, J., Frigge, M.L., Kong, A., Gulcher, J.R., Stefansson, K. et al. (2002) A susceptibility gene for late-onset idiopathic Parkinson's disease. Ann. Neurol., 52, 549–555.[CrossRef][Web of Science][Medline]

14. Chung, K.K., Zhang, Y., Lim, K.L., Tanaka, Y., Huang, H., Gao, J., Ross, C.A., Dawson, V.L. and Dawson, T.M. (2001) Parkin ubiquitinates the alpha-synuclein-interacting protein, synphilin-1: implications for Lewy-body formation in Parkinson disease. Nat. Med., 7, 1144–1150.[CrossRef][Web of Science][Medline]

15. Bandopadhyay, R., de Silva, R., Khan, N., Graham, E., Vaughan, J., Engelender, S., Ross, C., Morris, H., Morris, C., Wood, N.W. et al. (2001) No pathogenic mutations in the synphilin-1 gene in Parkinson's disease. Neurosci. Lett., 307, 125–127.[CrossRef][Web of Science][Medline]

16. Farrer, M., Destee, A., Levecque, C., Singleton, A., Engelender, S., Becquet, E., Mouroux, V., Richard, F., Defebvre, L., Crook, R. et al. (2001) Genetic analysis of synphilin-1 in familial Parkinson's disease. Neurobiol. Dis., 8, 317–323.[CrossRef][Web of Science][Medline]

17. O'Farrell, C., Pickford, F., Vink, L., McGowan, E. and Cookson, M.R. (2002) Sequence conservation between mouse and human synphilin-1. Neurosci. Lett., 322, 9–12.[CrossRef][Web of Science][Medline]

18. O'Farrell, C., Murphy, D.D., Petrucelli, L., Singleton, A.B., Hussey, J., Farrer, M., Hardy, J., Dickson, D.W. and Cookson, M.R. (2001) Transfected synphilin-1 forms cytoplasmic inclusions in HEK293 cells. Brain Res. Mol. Brain Res., 97, 94–102.[Medline]

19. Lee, G., Junn, E., Tanaka, M., Kim, Y.M. and Mouradian, M.M. (2002) Synphilin-1 degradation by the ubiquitin-proteasome pathway and effects on cell survival. J. Neurochem., 83, 346–352.[CrossRef][Web of Science][Medline]

20. Neystat, M., Rzhetskaya, M., Kholodilov, N. and Burke, R.E. (2002) Analysis of synphilin-1 and synuclein interactions by yeast two-hybrid beta-galactosidase liquid assay. Neurosci. Lett., 325, 119–123.[CrossRef][Web of Science][Medline]

21. Conway, K.A., Rochet, J.C., Bieganski, R.M. and Lansbury, P.T. Jr (2001) Kinetic stabilization of the alpha-synuclein protofibril by a dopamine-alpha-synuclein adduct. Science, 294, 1346–1349.[Abstract/Free Full Text]

22. Masliah, E., Rockenstein, E., Veinbergs, I., Mallory, M., Hashimoto, M., Takeda, A., Sagara, Y., Sisk, A. and Mucke, L. (2000) Dopaminergic loss and inclusion body formation in alpha-synuclein mice: implications for neurodegenerative disorders. Science, 287, 1265–1269.[Abstract/Free Full Text]

23. Lücking, C.B., Dürr, A., Bonifati, V., Vaughan, J., De Michele, G., Gasser, T., Harhangi, B.S., Meco, G., Denefle, P., Wood, N.W. et al. (2000) Association between early-onset Parkinson's disease and mutations in the parkin gene. French Parkinson's Disease Genetics Study Group. New Engl. J. Med., 342, 1560–1567.[Abstract/Free Full Text]

24. Tompkins, M.M., Basgall, E.J., Zamrini, E. and Hill, W.D. (1997) Apoptotic-like changes in Lewy-body-associated disorders and normal aging in substantia nigral neurons. Am. J. Pathol., 150, 119–131.[Abstract]

25. Berger, K., Hense, H.W., Rothdach, A., Weltermann, B. and Keil, U. (2000) A single question about prior stroke versus a stroke questionnaire to assess stroke prevalence in populations. Neuroepidemiology, 19, 245–257.[CrossRef][Web of Science][Medline]

26. Fahn, S. and Elton, R., Members of the UPDRS Development Committee (1987) Unified Parkinson's Disease Rating Scale. In Fahn, S., Marsden, C.D., Calne, D.B., Goldstein M. (eds), Recent Developments in Parkinson's Disease. Macmillan Health Care Information, Florham Park, NJ, pp. 211–128.

27. Miterski, B., Krüger, R., Wintermeyer, P. and Epplen, J.T. (2000) PCR/SSCP detects reliably and efficiently DNA sequence variations in large scale screening projects. Comb. Chem. High Throughput Screen., 3, 211–218.[Web of Science][Medline]

28. He, T.C., Zhou, S., da Costa, L.T., Yu, J., Kinzler, K.W. and Vogelstein, B. (1998) A simplified system for generating recombinant adenoviruses. Proc. Natl Acad. Sci. USA, 95, 2509–2514.[Abstract/Free Full Text]

CiteULike    Connotea    Del.icio.us    What's this?

This article has been cited by other articles:

				R. Rott, R. Szargel, J. Haskin, V. Shani, A. Shainskaya, I. Manov, E. Liani, E. Avraham, and S. Engelender Monoubiquitylation of {alpha}-Synuclein by Seven in Absentia Homolog (SIAH) Promotes Its Aggregation in Dopaminergic Cells J. Biol. Chem., February 8, 2008; 283(6): 3316 - 3328. [Abstract] [Full Text] [PDF]

				C. Klein and M. G. Schlossmacher Parkinson disease, 10 years after its genetic revolution: Multiple clues to a complex disorder Neurology, November 27, 2007; 69(22): 2093 - 2104. [Abstract] [Full Text] [PDF]

				F. P. Marx, A. S. Soehn, D. Berg, C. Melle, C. Schiesling, M. Lang, S. Kautzmann, K. M. Strauss, T. Franck, S. Engelender, et al. The proteasomal subunit S6 ATPase is a novel synphilin-1 interacting protein--implications for Parkinson's disease FASEB J, June 1, 2007; 21(8): 1759 - 1767. [Abstract] [Full Text] [PDF]

				D. Gherbassi, L. Bhatt, S. Thuret, and H. H. Simon Merging Mouse Transcriptome Analyses with Parkinson's Disease Linkage Studies DNA Res, May 23, 2007; (2007) dsm007v1. [Abstract] [Full Text] [PDF]

				E.-K. Tan and J. Jankovic Genetic testing in Parkinson disease: promises and pitfalls. Arch Neurol, September 1, 2006; 63(9): 1232 - 1237. [Full Text] [PDF]

				K. Tanji, T. Tanaka, F. Mori, K. Kito, H. Takahashi, K. Wakabayashi, and T. Kamitani NUB1 Suppresses the Formation of Lewy Body-Like Inclusions by Proteasomal Degradation of Synphilin-1 Am. J. Pathol., August 1, 2006; 169(2): 553 - 565. [Abstract] [Full Text] [PDF]

				E. Giaime, C. Sunyach, M. Herrant, S. Grosso, P. Auberger, P. J. McLean, F. Checler, and C. A. da Costa Caspase-3-derived C-terminal Product of Synphilin-1 Displays Antiapoptotic Function via Modulation of the p53-dependent Cell Death Pathway J. Biol. Chem., April 28, 2006; 281(17): 11515 - 11522. [Abstract] [Full Text] [PDF]

				A. Eyal, R. Szargel, E. Avraham, E. Liani, J. Haskin, R. Rott, and S. Engelender Synphilin-1A: An aggregation-prone isoform of synphilin-1 that causes neuronal death and is present in aggregates from {alpha}-synucleinopathy patients PNAS, April 11, 2006; 103(15): 5917 - 5922. [Abstract] [Full Text] [PDF]

				E. Avraham, R. Szargel, A. Eyal, R. Rott, and S. Engelender Glycogen Synthase Kinase 3beta Modulates Synphilin-1 Ubiquitylation and Cellular Inclusion Formation by SIAH: IMPLICATIONS FOR PROTEASOMAL FUNCTION AND LEWY BODY FORMATION J. Biol. Chem., December 30, 2005; 280(52): 42877 - 42886. [Abstract] [Full Text] [PDF]

				K. L. Lim, K. C. M. Chew, J. M. M. Tan, C. Wang, K. K. K. Chung, Y. Zhang, Y. Tanaka, W. Smith, S. Engelender, C. A. Ross, et al. Parkin Mediates Nonclassical, Proteasomal-Independent Ubiquitination of Synphilin-1: Implications for Lewy Body Formation J. Neurosci., February 23, 2005; 25(8): 2002 - 2009. [Abstract] [Full Text] [PDF]

				H. Ohtake, P. Limprasert, Y. Fan, O. Onodera, A. Kakita, H. Takahashi, L. T. Bonner, D. W. Tsuang, I. V.J. Murray, V. M.-Y. Lee, et al. {beta}-Synuclein gene alterations in dementia with Lewy bodies Neurology, September 14, 2004; 63(5): 805 - 811. [Abstract] [Full Text] [PDF]

				E. Liani, A. Eyal, E. Avraham, R. Shemer, R. Szargel, D. Berg, A. Bornemann, O. Riess, C. A. Ross, R. Rott, et al. Ubiquitylation of synphilin-1 and {alpha}-synuclein by SIAH and its presence in cellular inclusions and Lewy bodies imply a role in Parkinson's disease PNAS, April 13, 2004; 101(15): 5500 - 5505. [Abstract] [Full Text] [PDF]

				K. Hedrich, A. Djarmati, N. Schafer, R. Hering, C. Wellenbrock, P. H. Weiss, R. Hilker, P. Vieregge, L. J. Ozelius, P. Heutink, et al. DJ-1 (PARK7) mutations are less frequent than Parkin (PARK2) mutations in early-onset Parkinson disease Neurology, February 10, 2004; 62(3): 389 - 394. [Abstract] [Full Text] [PDF]

				M. Tanaka, Y. M. Kim, G. Lee, E. Junn, T. Iwatsubo, and M. M. Mouradian Aggresomes Formed by {alpha}-Synuclein and Synphilin-1 Are Cytoprotective J. Biol. Chem., February 6, 2004; 279(6): 4625 - 4631. [Abstract] [Full Text] [PDF]

				Y. Nagano, H. Yamashita, T. Takahashi, S. Kishida, T. Nakamura, E. Iseki, N. Hattori, Y. Mizuno, A. Kikuchi, and M. Matsumoto Siah-1 Facilitates Ubiquitination and Degradation of Synphilin-1 J. Biol. Chem., December 19, 2003; 278(51): 51504 - 51514. [Abstract] [Full Text] [PDF]

				T. M. Dawson and V. L. Dawson Molecular Pathways of Neurodegeneration in Parkinson's Disease Science, October 31, 2003; 302(5646): 819 - 822. [Abstract] [Full Text] [PDF]

				N. Pankratz, W. C. Nichols, S. K. Uniacke, C. Halter, J. Murrell, A. Rudolph, C. W. Shults, P. M. Conneally, T. Foroud, and the Parkinson Study Group Genome-wide linkage analysis and evidence of gene-by-gene interactions in a sample of 362 multiplex Parkinson disease families Hum. Mol. Genet., October 16, 2003; 12(20): 2599 - 2608. [Abstract] [Full Text] [PDF]

				O. CHIBA-FALEK and R.L. NUSSBAUM Regulation of {alpha}-Synuclein Expression: Implications for Parkinson's Disease Cold Spring Harb Symp Quant Biol, January 1, 2003; 68(0): 409 - 416. [Abstract] [PDF]

This Article Abstract FREE Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Search for citing articles in: ISI Web of Science (66) Request Permissions Google Scholar Articles by Marx, F. P. Articles by Krüger, R. Search for Related Content PubMed PubMed Citation Articles by Marx, F. P. Articles by Krüger, R. Social Bookmarking          What's this?

Online ISSN 1460-2083 - Print ISSN 0964-6906

Copyright © 2009 Oxford University Press

Oxford Journals Oxford University Press

• Site Map
• Privacy Policy
• Frequently Asked Questions

Other Oxford University Press sites: Oxford University Press Oxford Journals China Oxford Journals Japan American National Biography Booksellers' Information Service Children's Fiction and Poetry Children's Reference Corporate & Special Sales Dictionaries Dictionary of National Biography Digital Reference English Language Teaching Higher Education Textbooks Humanities International Education Unit Law Medicine Music Online Products Oxford English Dictionary Reference Rights and Permissions Science School Books Social Sciences Very Short Introductions World's Classics