J Biol Chem, Vol. 274, Issue 48, 33855-33858, November 26, 1999

COMMUNICATION
Degradation of -Synuclein by Proteasome^*

M. Catherine Bennett, John F. Bishop, Yan Leng, P. Boon Chock^§, Thomas N. Chase, and M. Maral Mouradian^¶

From the  Experimental Therapeutics Branch, NINDS, and the ^§ Laboratory of Biochemistry, NHLBI, National Institutes of Health, Bethesda, Maryland 20892

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Mutations in -synuclein are known to be associated with Parkinson's disease (PD). The coexistence of this neuronal protein with ubiquitin and proteasome subunits in Lewy bodies in sporadic disease suggests that alterations of -synuclein catabolism may contribute to the pathogenesis of PD. The degradation pathway of -synuclein has not been identified nor has the kinetics of this process been described. We investigated the degradation kinetics of both wild-type and A53T mutant 6XHis-tagged -synuclein in transiently transfected SH-SY5Y cells. Degradation of both isoforms followed first-order kinetics over 24 h as monitored by the pulse-chase method. However, the t_1/2 of mutant -synuclein was 50% longer than that of the wild-type protein (p < 0.01). The degradation of both recombinant proteins and endogenous -synuclein in these cells was blocked by the selective proteasome inhibitor -lactone (40 µM), indicating that both wild-type and A53T mutant -synuclein are degraded by the ubiquitin-proteasome pathway. The slower degradation of mutant -synuclein provides a kinetic basis for its intracellular accumulation, thus favoring its aggregation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Converging lines of evidence link the neuronal protein -synuclein to the pathogenesis of Parkinson's disease (PD).¹ Two different point mutations in -synuclein, A53T (1) and A30P (2) have been identified in separate families with dominantly transmitted PD. Although -synuclein gene mutations linked to hereditary PD are rare, the associated disease pathology is indistinguishable from that of sporadic cases. Thus, these mutations may point to a more general involvement of -synuclein in the pathogenesis of PD.

-Synuclein is the major component of Lewy bodies found in the brains of sporadic PD patients, suggesting that abnormal processing of -synuclein is a common feature of this disease (3-5). In addition, Lewy bodies contain abundant ubiquitin (6, 7-9). Protein polyubiquitination is the marker for degradation by proteasome, the proteolytic complex that degrades many cytoplasmic proteins (10, 11). Furthermore, proteasome subunits have been identified in Lewy bodies (12). Taken together, these immunohistochemical features indicate that -synuclein catabolism may be impaired in PD, causing its accumulation in the cytoplasm and promoting its aggregation into Lewy bodies. The mechanism of the concomitant neurodegeneration is presently unknown.

The kinetics of -synuclein turnover have not been determined, nor has its degradation pathway been identified. In the present investigation, we studied the rate of catabolism for both recombinant wild-type and A53T mutant forms of -synuclein in transiently transfected cells of the human dopaminergic neuroblastoma cell line SH-SY5Y (13). We also tested the hypothesis that -synuclein is catabolized by proteasome using the selective proteasome inhibitor -lactone.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Plasmid Construction-- Human brain RNA (CLONTECH) was subjected to reverse transcriptase-PCR utilizing the following primers complementary to the human -synuclein coding region: h-syn RT, nt 464-448 relative to the translation start codon (5'-ATCTGTCAGCAGATCTC-3'); h-syn S, nt 1-26 (5'-ATGGATGTATTCATGAAAGGACTTTC-3'); h-syn AS, nt 422-399 (5'-TTAGGCTTCAGGTTCGTAGTCTTG-3'). The resultant PCR product was subcloned into pCR2.1 (Invitrogen) and subjected to site-directed mutagenesis (QuikChange, Stratagene) to generate -synuclein cDNA containing a single base substitution (G to A) at nucleotide 157. These pCR2.1 constructs were then used as templates to add 5' KpnI and 3' XbaI sites to both wild-type and mutant -synuclein sequences by PCR using -synuclein-specific primers that were 3'-extended with the appropriate restriction endonuclease target. After digesting the resultant PCR products with KpnI and XbaI, they were ligated into the respective sites of pcDNA3.1/His-A (Invitrogen), generating pcDNA3.1/5'-His-syn-wt and pcDNA3.1/5'-His-syn-mut, which express wild-type and A53T mutant forms of -synuclein, respectively, fused to the His-tag at their N termini. The orientation and sequence of the inserts were verified by DNA sequencing. Plasmids were purified using a commercial plasmid purification system (Qiagen).

Cell Culture and Transfection-- Human dopaminergic neuroblastoma SH-SY5Y cells (ATCC) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and were kept at 37 °C in humidified 10% CO₂/90% air. Cells were grown in 100-mm dishes to 80-90% confluence and transiently transfected with 8 µg/dish of either pcDNA3.1/5'-His-syn-wt or pcDNA3.1/5'-His-syn-mut, using Pfx-6 (Invitrogen) according to the supplier's recommended procedure. As a control, -galactosidase was expressed in cells transfected with pcDNA3.1(+)Myc-His/LacZ (Invitrogen).

[³⁵S]Methionine Pulse-Chase Experiments-- The day following transfection, cells were rinsed with 5 ml of Dulbecco's phosphate-buffered saline (DPBS) and incubated for 4-6 h in 5 ml of Met/Leu-free DMEM containing 100 µCi of [³⁵S]Met Trans-labeled methionine (ICN). Cells were then rinsed with 5 ml of DPBS and incubated for chase intervals of 0, 2, 4, 8, and 24 h in 5 ml of non-radioactive DMEM supplemented with 10% fetal bovine serum. Three independent experiments were performed in quadruplicate.

Proteasome Inhibition by -Lactone-- SH-SY5Y cells transiently expressing either wild-type or mutant His-tagged -synuclein fusion proteins were labeled with [³⁵S]methionine and then incubated for 4 h with unlabeled medium containing 40 µM -lactone (Boston Biochem Inc.) or vehicle (DMSO, final concentration 0.1%). This concentration of -lactone was chosen to maximally inhibit proteasome function without inducing nonspecific cell toxicity (14). Three independent experiments were carried out using 3-4 plates for each condition. Non-transfected SH-SY5Y cells were also incubated in culture medium containing 40 µM -lactone or DMSO for up to 4 h, to determine the effect of proteasome inhibition on endogenous -synuclein.

Immunoprecipitation and Protein Detection-- At the end of chase intervals, cells were washed with 5 ml of ice-cold DPBS, collected in microcentrifuge tubes by scraping in 1 ml of DPBS, and centrifuged for 2 min at 3000 × g. Cells were then suspended in 500 ml of RIPA buffer (150 mM NaCl, 50 mM Tris-Cl, pH 7.4, 0.25% w/v sodium deoxycholate, 0.1% v/v Nonidet P-40, 100 µM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 10 µg/ml leupeptin) and lysed by triturating 10-12 times. The resultant cell lysate was centrifuged for 10 min at 16,000 × g, and the supernatant was transferred to a microcentrifuge tube pretreated with a blocking agent (SuperBlock, Pierce). Following the addition of 10 µl of 6XHis monoclonal antibody (1:500, CLONTECH), tubes were left to shake at 1000 Hz overnight at 4 °C. The antibody-antigen complex was extracted from the lysate by incubating with 20 µl of protein A-Sepharose 4B (Zymed Laboratories Inc.) for 1 h while shaking, followed by centrifugation for 1 min at 10,000 × g. Protein A-Sepharose pellets were resuspended in RIPA buffer and washed three times, taking care to remove all RIPA buffer after a two-stage final spin. Proteins were denatured with 10 µl of 4× SDS buffer (Novex) containing 10% (v/v) 2--mercaptoethanol and heated at 85-90 °C for 5 min, after which samples were centrifuged at 16,000 × g for 3 min. Samples (10 µl/lane) and prestained molecular weight markers (SeeBlue, Novex) were electrophoresed in 4-20% SDS-polyacrylamide gels, rinsed, and vacuum-dried. Radiolabeled -synuclein bands were visualized and quantified by PhosphorImager analysis (Molecular Dynamics). Endogenous -synuclein in non-transfected cells was detected in lysates by Western blot analysis using a monoclonal antibody to -synuclein (Transduction Laboratories) with chemiluminescence (Amersham Pharmacia Biotech), and signals were quantified by optical densitometry using NIH Image 1.61.

Statistical Analyses-- In pulse-chase experiments, the decay of [³⁵S]--synuclein was followed over 24 h. A one-phase exponential decay model (A · e^kt + B) was fit to each data set using the nonlinear regression analysis program of GraphPad Prism 2.0 (GraphPad Software). The goodness-of-fit of each data set to its best-fit theoretical curve was assessed as the square of the correlation coefficient (R²). Best-fit decay constants for the two -synuclein isoforms were compared by t tests. In -lactone experiments, groups were compared by one-way analysis of variance followed by the Tukey post hoc Multiple Comparison Test (GraphPad Software). To permit quantitative comparison of -synuclein decay across experimental replications, data were normalized as a percentage of the mean zero-hour label and expressed as percent ± S.E. However, the variance within each set of raw data was preserved for statistical calculations.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Wild-type and A53T mutant isoforms of 6XHis-tagged human -synuclein fusion proteins were expressed in the human neuroblastoma cell line SH-SY5Y by transient transfections. Following labeling with [³⁵S]methionine, these recombinant proteins were immunoprecipitated with a monoclonal antibody to the 6XHis-tag and detected by SDS-PAGE as a single band migrating with an apparent molecular mass of approximately 21 kDa (Fig. 1). The migration of these fusion proteins, which include 20 additional amino acids forming the tag, is consistent with previous reports that unconjugated -synuclein monomer migrates with an apparent molecular mass of 19 kDa (15).

View larger version (71K): [in this window] [in a new window]   Fig. 1.   [35S]Met-labeled recombinant proteins. Autoradiograph of SDS-polyacrylamide gel showing wild-type and A53T mutant His-tagged -synuclein transiently expressed in SH-SY5Y cells. 6XHis-tagged -galactosidase (-gal) was expressed as a control. Arrows point to the specific recombinant proteins in the respective transfectants. Molecular mass markers are shown in the left lane.

The degradation rates of both isoforms of [³⁵S]Met-labeled His-tagged -synuclein were studied in pulse-chase experiments using chase intervals of 0-24 h (Fig. 2 and Table I). The decay of both isoforms of -synuclein correlated highly with one-phase exponential equations fit to each data set, with R² = 0.9362 for wild-type -synuclein and R² = 0.9286 for the mutant protein, implying a single rate-limiting step for the degradation of approximately 70% of each protein. However, almost 30% of the original -synuclein label consistently remained at the 24-hour time point, long after the initial decay had reached its apparent plateau (the variable "B" of the decay equation). Fraction B could be the result of an initially insoluble form of -synuclein that aggregated because of overexpression and was then redissolved by the denaturation process (16). Alternatively, another pool of -synuclein undergoing a slower turnover cannot be excluded. The goodness-of-fit of the 24-hour degradation data to a one-phase exponential model demonstrates that the early and major process of -synuclein catabolism obeys first order kinetics.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Degradation kinetics of -synuclein. The amount of residual 35S-labeled His-tagged wild-type () and A53T mutant -synuclein () in SH-SY5Y cells incubated in unlabeled chase medium for intervals of up to 24 h, expressed as percent (±S.E.) of the label measured immediately after pulse labeling (0 h). The two curves represent each data set fit to a first order decay equation. The two horizontal broken lines correspond to the B-constants, which represent the residual protein at the asymptote in the decay equations obtained for each isoform. The initial value of the -synuclein label, normalized to 100%, represents the sum of the decay span A and B. The t1/2 for each decay curve corresponds to the midpoint of the amplitude of A (shown by vertical lines) rather than to the time point when residual -synuclein equals 50% of its initial signal.

View this table: [in this window] [in a new window]   Table I Degradation kinetics of -synuclein Wild-type and mutant recombinant -synuclein degradation data were fit to a first-order decay function: f(t) = A · ekt + B, where A is the Span or total range of protein decay over the measured interval, B is the Plateau or residual protein label at the decay asymptote, k is the decay rate constant, and t is the duration of the chase incubation. t1/2 (expressed in hours) is calculated as 0.693/k. Values are expressed as mean ± S.E. The half-lives of wild-type and mutant proteins are significantly different, while other decay constants are not. The square of the correlation coefficient R2 is a measure of goodness-of-fit.

The estimated half-lives calculated for wild-type and mutant isoforms of His-tagged -synuclein were consistently and significantly different. Although the t_1/2 for wild-type was 1.84 ± 0.16 h, that of the mutant form was 2.76 ± 0.24 h (p < 0.01), representing a 50% prolongation. The overexpressed recombinant proteins in this experimental paradigm cannot be assumed to have the same half-lives as their endogenous counterparts because synthesis rate can affect degradation rate (17, 18). Nevertheless, the finding that mutant -synuclein degrades more slowly than the wild-type under the same experimental conditions suggests a potential mechanism whereby the mutated protein accumulates in the cytoplasm at higher concentrations. This observation in a cellular system, along with the higher propensity of A53T mutant -synuclein to aggregate into filamentous structures in vitro (19-21), reveals a possible physicochemical basis for the pathogenesis of some forms of PD.

The specific and irreversible proteasome inhibitor -lactone significantly inhibited the degradation of both wild-type and mutant His-tagged -synuclein (Fig. 3). The amount of both of the labeled -synuclein isoforms in cells incubated in the control chase medium significantly declined by 4 h. By contrast, no significant decrease of either -synuclein isoform was found in cells incubated with -lactone. Similarly, steady state levels of -synuclein in non-transfected SH-SY5Y cells were increased after -lactone treatment compared with vehicle (Fig. 4), indicating that the endogenous protein is also catabolized by proteasome. The lack of detection of polyubiquitin laddering in our experiments is likely due to concurrent de-ubiquitination by isopeptidases, which often cause an increase in non-ubiquitinated substrate, rather than an accumulation of ubiquitinated species, to be the more conspicuous effect of proteasome inhibition (22). Substrate polyubiquitination is required prior to the degradation of most proteins known to be processed by the eucaryotic proteasome (23). Taken together, the findings that -lactone blocks degradation of endogenous -synuclein and both recombinant isoforms provide strong evidence that this protein is degraded in vivo by the ubiquitin-proteasome proteolytic pathway.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Inhibition of -synuclein degradation by -lactone. A, representative autoradiographs of 35S-labeled wild-type and A53T mutant His-tagged -synuclein transiently expressed in SH-SY5Y cells after a 4-h incubation in unlabeled medium in the presence of 40 µM -lactone or 0.1% DMSO. B, data combined from all experiments were normalized as described under "Experimental Procedures" and expressed as the mean ± S.E. Wild-type: a, p < 0.001 compared with 0 h and with 4 h + -lactone; Mutant: b, p < 0.001 compared with 0 h; and c, p < 0.01 compared with 4 h + -lactone. No significant differences were found between the 0 h and the 4 h + -lactone values for either wild-type or mutant protein.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Inhibition of endogenous -synuclein degradation by -lactone. Endogenous -synuclein in SH-SY5Y cells detected by immunoblotting followed by chemiluminescence was increased by 79, 61, and 52% after incubation with 40 µM -lactone for 1, 2, and 4 h, respectively, compared with that found in cells incubated with vehicle (0.1% DMSO) for 4 h.

In the few kindreds in which the A53T -synuclein mutation has been found, all individuals identified as carriers of this mutation have developed PD, with symptom onset approximately a decade earlier than average for the sporadic disease. The present finding of a significantly longer half-life of the A53T mutant relative to wild-type -synuclein suggests that the mutant is degraded less efficiently by proteasome. Even a minor impediment to -synuclein catabolism could shift the cellular equilibrium toward an increase in cytosolic concentration, thus increasing the opportunity for aggregation. The biochemistry of proteasome catabolism is complex and involves many protein-targeting reactions in addition to proteasome proteolysis. The kinetics of catabolism can be altered at multiple steps in the proteasome pathway. Subtle alterations in the kinetics of these reactions could produce small changes in the cellular biochemistry that could have a cumulative effect over decades and may underlie slowly progressing neurodegenerative disorders such as PD.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: NINDS, National Institutes of Health, 10 Center Dr., MSC 1406, Bldg. 10, Rm. 5C-116, Bethesda, MD 20892-1406. Tel.: 301-496-7872; Fax: 301-496-6609; E-mail: mmm@helix.nih.gov.

	ABBREVIATIONS

The abbreviations used are: PD, Parkinson's disease; PCR, polymerase chain reaction; nt, nucleotide(s); h-syn, human -synuclein; DMEM, Dulbecco's modified Eagle's medium; DPBS, Dulbecco's phosphate-buffered saline; DMSO, dimethylsulfoxide.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES