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Direct binding and functional coupling of -synuclein to the dopamine transporters accelerate dopamine-induced apoptosis

FRANK J. S. LEE^*,1, FANG LIU^,, ZDENEK B. PRISTUPA^, and HYMAN B. NIZNIK^*,,,

^* Department of Pharmacology,
Psychiatry and
Institute of Medical Sciences, University of Toronto, Toronto, Ontario M5S 1A8, Canada; and
Lab of Molecular Neurobiology, Centre for Addiction and Mental Health, Toronto, Ontario, M5T 1R8, Canada

¹Correspondence: Laboratory of Molecular Neurobiology, Centre for Addiction and Mental Health, 250 College St., Toronto, Ontario, M5T 1R8 Canada. E-mail: f.lee{at}utoronto.ca

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Mutations in -synuclein, a protein highly enriched in presynaptic terminals, have been implicated in the expression of familial forms of Parkinson’s disease (PD) whereas native -synuclein is a major component of intraneuronal inclusion bodies characteristic of PD and other neurodegenerative disorders. Although overexpression of human -synuclein induces dopaminergic nerve terminal degeneration, the molecular mechanism by which -synuclein contributes to the degeneration of these pathways remains enigmatic. We report here that -synuclein complexes with the presynaptic human dopamine transporter (hDAT) in both neurons and cotransfected cells through the direct binding of the non-Aß amyloid component of -synuclein to the carboxyl-terminal tail of the hDAT. -Synuclein–hDAT complex formation facilitates the membrane clustering of the DAT, thereby accelerating cellular dopamine uptake and dopamine-induced cellular apoptosis. Since the selective vulnerability of dopaminergic neurons in PD has been ascribed in part to oxidative stress as a result of the cellular overaccumulation of dopamine or dopamine-like molecules by the presynaptic DAT, these data provide mechanistic insight into the mode by which the activity of these two proteins may give rise to this process.—Lee, F. J. S., Liu, F., Pristupa, Z. B., Niznik, H. B. Direct binding and functional coupling of -synuclein to the dopamine transporter accelerate dopamine-induced apoptosis.

Key Words: DA uptake • cell death • coimmunoprecipitation • GST fusion proteins • Parkinson’s disease

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
RECENT INTEREST IN the functional role of -synuclein has peaked with the observation that mutations in -synuclein are linked in some familial cases of Parkinson’s disease (PD) (1 2 3) , characterized by the preferential and progressive destruction of nigrostriatal dopaminergic neurons (4 5 6) . Moreover, studies have shown the accumulation of -synuclein in dystrophic neurons and Lewy bodies of idiopathic Parkinson’s disease and other neurodegenerative disorders (7 8 9 10 11) .

-Synuclein is one member of the synuclein gene family that is abundantly expressed in various brain regions and is highly enriched in presynaptic terminals (12 13 14 15) . Structurally, -synuclein is composed of three modular domains, including an amino-terminal lipid binding -helix, a ß-amyloid binding domain that encodes the non-Aß component (NAC) of Alzheimer’s diseased plaques, and a carboxyl-terminal acidic tail. The structure of -synuclein allows the molecule to exist in either a random or a natively unfolded conformation or as an alpha helix in the presence of phospholipids (16 , 17) , suggesting a highly dynamic regulation of -synuclein function depending on the local cellular milieu. Indeed, recent studies have suggested various cellular roles for -synuclein that include possible modifications of membrane and cell surface signaling events (12) . -Synuclein is thought to play a role in vesicle function and has been shown to inhibit phospholipase D2 activity and production of phosphatidic acid (18) . Additional evidence suggests the involvement of -synuclein in the protein ubiquitination process (19) , modulation of 14–3-3 chaperone molecules (20) , and as a possible substrate and/or inhibitor of protein kinase-dependent pathways (21) .

The overall structure and presynaptic localization of -synuclein coupled with its potential role in modifying synaptic function, as exhibited by the impairment of nigrostriatal dopaminergic neurotransmission in transgenic -synuclein deficient (-syn^-/-) mice (22) , raised the possibility that -synuclein may act as a functional binding partner for another dopaminergic presynaptic protein, the dopamine transporter (DAT). The DAT is a member of a large gene family of Na⁺- and Cl^--dependent transporters with a common topology of 12 putative transmembrane (TM) domains, intracellular amino and carboxyl termini, a large extracellular loop between TM 3 and 4 containing numerous consensus sequences for N-linked glycosylation, and putative sites for phosphorylation by protein kinase A (PKA), PKC, and CaM kinase II within putative intracellular domains including the amino and carboxyl termini. The DAT is expressed in presynaptic terminals of substantia nigral neurons, where it mediates the reuptake of synaptically released dopamine (DA) (23 24 25) .

DA has been documented to induce neurotoxicity (26 , 27) . The mechanism by which DA induces its neurotoxic effects is generally linked to oxidative metabolism, which releases reactive oxygen species, free radicals, and quinones, causing damage to cellular protein, lipid, and DNA elements (28) . Furthermore, the DAT mediates the reuptake of MPTP, a neurotoxin that induces parkinsonian symptoms of affected individuals (29) . Therefore, it is conceivable that enhanced DAT activity would not only facilitate increases in intracellular content of DA or DA-like molecules, but may also bolster the production of reactive free radical metabolites that would induce the neurodegeneration of dopaminergic cells. We now report the functional effects of a direct interaction between two seemingly unrelated proteins: -synuclein and the DAT.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cloning the -synuclein transcript and cell transfection
The cDNA transcript of -synuclein was amplified via PCR from a human cDNA substantia nigra (Clontech, Palo Alto, Calif.) library using materials and methods previously described (30) . The 5' and 3' oligonucleotides [SYN-_N-CGG GGT ACC GCC GCC ACC ATG GAT GTG TTC ATG AA; SYN_C-CCG CTC GAG TTC TTA GGC CTC AGG TTC GTA] incorporated KpnI and XhoI restriction sites (boldface), respectively, to facilitate subcloning into pcDNA3. The subcloned cDNA insert was resequenced to detect any spurious PCR-generated errors. Deduced amino acid sequence was identical to that reported in GenBank accession #L08850. hDAT and -synuclein cDNA constructs inserted into pcDNA3 were used to transiently transfect Ltk^- cells by the DEAE-dextran. All other hDAT mutant constructs were as described (37) . Two to 4 days after transfection with 20 µg of plasmid, cDNA cells were used for [³H]DA uptake, [³H]CFT binding, or confocal microscopy analysis as described previously (30 , 31) .

Yeast two-hybrid screening and ß-galactosidase assays
PCR was used to generate -synuclein-activating domain (AD) fusion constructs in either pACT2 or pGADT7 and binding domain (BD) fusion constructs with the hDAT intracellular amino-terminal (NT: Met¹-Lys⁶⁶), carboxyl-terminal (CT1: Leu⁵⁸³-Val ⁶²⁰), and carboxyl-terminal fragments (CT2: Glu⁵⁹⁸-Val⁶²⁰; CT3: Leu⁵⁸³-Pro⁵⁹⁷) in pAS2 or pGBKT7. Appropriate pairs of AD and BD constructs were cotransformed into the yeast strain Y187 grown at 30°C for 3 days on Leu^-/Trp^- medium lacking leucine and tryptophan, followed by assay of lacZ reporter activation, present downstream of a GAL4 binding sequence in Y187, as described by the manufacturer (Clontech). All control transformations were negative for ß-galactosidase activity except for BD/p53 with AD/T antigen, used as a positive control. All constructs were sequenced to determine appropriate splice fusion and absence of spurious PCR-generated mutations.

Affinity purification (‘pull-down’), coimmunoprecipitation, and Western blotting
-Synuclein GST fusion proteins and various hDAT mutant constructs were constructed via PCR as described previously (32) . 5' and 3' oligonucleotides were directed to specific areas of cDNA constructs and contained restriction endonuclease sites to facilitate subcloning into pGEX2T, pGEX4T-3, or pcDNA3. GST fusion protein constructs incorporated stop codons in the 3'oligonucleotide whereas minigene constructs incorporated initiation methionines. GST fusion proteins were prepared from bacterial lysates as described by the manufacturer (Pharmacia, Uppsala, Sweden). All constructs were resequenced to detect any spurious PCR-generated errors. Coimmunoprecipitation and GST affinity pull-down experiments were carried out as described previously with minor modifications (31) . Rat striata (200–300 mg) or transfected Ltk^- cells (2x10⁷) were homogenized in buffer (50 mM Tris pH 7.5, 120 mM NaCl, 1.5 mM CaCl₂, 5 mM MgCl₂, 5 mM KCl, 5 mM EDTA) with a protease inhibitor mixture (Sigma, St. Louis, Mo.; 1 ml/20 g of tissue). Homogenates were solubilized with 1% digitonin, followed by the addition of secondary solubilization buffer (50 mM Tris, pH 7.6, 150 mM NaCl, 1% igepalCa630, 0.5% sodium deoxycholate, 2 mM EDTA, 1 mM sodium orthovanadate, 1 mM PMSF, and 1% Triton X-100), and centrifuged at 10,000 g at 4°C for 15 min. To the solubilized extracts (1–1.5 mg) was added DAT mAb (1:100), -synuclein polyclonal Ab (1:100; Chemicon, El Segundo, Calif.), or rabbit sera ( 5 µg) 4–12 h at 4°C, followed by 50 µl of either protein A-agarose (Bio-Rad, Hercules, Calif.) or anti-rat IgG-agarose (Sigma) for 12 h at 4°C. Pellets were washed three times in buffer, resuspended in SDS sample buffer, and subjected to SDS-PAGE. For GST affinity precipitation experiments, solubilized striatal extracts were incubated with GST fusion proteins (50–75 µg), followed by incubation with glutathione Sepharose beads for 1 h at room temperature. Beads were washed three times with buffer and eluted twice with 25 µl glutathione elution buffer. Eluates were incubated with SDS sample buffer and subjected to SDS-PAGE. Blots were blocked with 5% nonfat dried milk dissolved in TBST (10 mM Tris, 150 mM NaCl, and 0.1% Tween-20) for 1 h at room temperature and incubated with primary antibodies for either DAT (1:1000) or -synuclein (1:1000, Chemicon) for 10 h at 4°C. Proteins were then visualized using peroxidase-conjugated secondary antibodies (Sigma) and enhanced chemiluminescence (Amersham, Arlington Heights, Ill.).

[³H]dopamine uptake analysis
We measured dopamine uptake on intact cells as described previously (30) . Two to 4 days after transfection in 24-well plates (2x10⁵ cells seeded per well), medium was removed and wells were rinsed with 0.5 ml of uptake buffer (5 mM Tris, 7.5 mM HEPES, 120 mM NaCl, 5.4 mM KCl, 1.2 mM CaCl₂, 1.2 mM MgSO₄, 1 mM ascorbic acid, 5 mM glucose, pH 7.1). Cells were then preincubated in duplicate with the indicated concentrations of dopaminergic agents (10^-13 to 10^-4 M) 5 min before the addition of 0.25 ml of 20 nM [³H]dopamine (final concentration) and incubated for 10 min at room temperature in a total volume of 0.5 ml. Nonspecific [³H]dopamine (37–53 Ci/mmol) uptake was defined in the presence of 10 µM mazindol. Wells were rinsed twice with 0.5 ml of uptake buffer; cells were solubilized in 0.5 ml of 1% SDS and collected to measure incorporated radioactivity using a Beckman liquid scintillation counter (LS 6000SC).

[³H]CFT binding
Measurement of [³H]CFT binding was performed on intact cells as previously reported (30) under conditions similar to those described above. Medium was removed and cells were rinsed with 0.5 ml of buffer, then incubated with 0.25 ml of buffer or drug for 5 min before the addition of 0.25 ml [³H]CFT (4 nM final concentration). After a 2 to 3 h incubation at 4°C, cells were washed twice with 0.5 ml of ice-cold buffer, solubilized with 1% SDS, and bound radioligand was measured for radioactivity as described above. Most competition assays were performed using 12 different concentrations (in duplicate) of the drug. Nonspecific binding was determined in the presence of 10 µM mazindol.

For all experiments, direct assay comparisons between cotransfections and single transfections were conducted in parallel, using the same dilutions of drug, on the same batch of transfected cells.

Cell culture and confocal imaging
Human progenitor dopaminergic cells were differentiated and maintained as described by the manufacturer (Clonexpress, Inc., Gaithersburg, Md.) and cultured for 1–2 wk before use. For double-label immunoconfocal microscopy, DAT mAbs and -synuclein polyclonal Ab (Chemicon) were used as primary antibodies in overnight incubations at 4°C and detected using secondary antibodies (Sigma) conjugated to FITC or TRITC fluorophores (31) . Control cells in which no primary Ab was included did not reveal any immunoreactive staining.

Cell transfection, apoptosis detection, and fluorescence microscopy
Human embryonic kidney 293 (HEK293) cells were transfected through a calcium-phosphate method with 15 µg of hDAT-pcDNA3 alone or combined with 15 µg of -synuclein-pcDNA3 and, where indicated, with 15 µg of DAT-CT^583–652-pcD. Two or 3 days after transfection, cells were either treated or untreated for 5 h with 50 µM DA (in -MEM, containing 1% FBS). After induction of apoptosis, cells were washed (2x) with prewarmed -MEM and incubated with the ApoAlert MitoSensor reagent for 20 min at 37°C, as described by the manufacturer (Clontech). Cells were subsequently washed once with -MEM and examined under a fluorescence microscope (Leica DM1RB) with a computer image capture capability. The proportion of healthy (orange-red fluorescence) to apoptotic (green fluorescence) cells was quantified using image capture software (MCID 5.1, Imaging Research Inc., St. Catharine’s, Ontario, Canada) with the capacity for automatic target detection, identifying fluorescing images through defined optical density and spatial criteria parameters.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Characterization of the hDAT--synuclein interaction
To assess potential direct protein–protein interactions between -synuclein and the human DAT, we incorporated divergent structural domains of the hDAT into DNA binding domain of a yeast two hybrid system (Clontech) and examined their ability to interact with the DNA-activating domains expressing full-length -synuclein cloned from the human substantia nigra. As illustrated in Fig. 1a , only fusion hybrids encoding the intracellular CT tail domain of DAT displayed direct functional binding interactions with -synuclein. This binding was specific since sequence domains encoded by the intracellular amino-terminal of hDAT did not recognize motifs on -synuclein. To define critical amino acids of the hDAT-CT tail that confer putative binding interactions with -synuclein, we assessed the ability of the amino vs. carboxyl-terminal portion of the CT tail to interact with -synuclein in this system. As depicted in Fig. 1a , -synuclein and hDAT complex formation appears dependent only on interaction with the last 22 amino acids of the CT tail of hDAT [Glu⁵⁹⁸-Val⁶²⁰] (DAT-CT2) and not on sequence motifs encoded within the first 15 amino acids [Leu⁵⁸³-Pro⁵⁹⁷] (DAT-CT1). These data suggest that hDAT--synuclein complex formation is mediated via direct protein–protein binding interactions between distal amino acid sequence motifs of the hDAT-CT tail with -synuclein and is not a product of indirect interactions with either unidentified dopamine transporter accessory proteins or known -synuclein binding partners (33 , 34) .

View larger version (42K): [in this window] [in a new window]   Figure 1. Association of -synuclein with hDAT. a) Yeast 2-hybrid system depicting direct interaction of the indicated intracellular fragments of hDAT as DNA binding domain (BD) hybrids with full-length -synuclein-activating domains (AD). Transporter fragments include DAT-NT, containing the amino terminus up to the first transmembrane domain (TM1); DAT-CT1, containing the entire carboxyl terminus after the 12th transmembrane domain (TM12); DAT-CT2, encoding amino acids 598 to 620, the distal end of the carboxyl terminus; and DAT-CT3, encoding amino acids 583 to 597, the carboxyl tail region proximal to TM12. Combinations of AD and BD plasmids were analyzed for activation of the lacZ reporter gene when coexpressed in the yeast strain Y187, with positive interactions apparent within 3–6 h. No interaction was observed between synuclein-AD constructs and Gal4 BD fused to murine p53 or human lamina C or between any of the DAT-BD fusions and Gal4 AD fused to SV40 large T antigen. b) DAT coimmunoprecipitated with -synuclein antibodies from rat striatal extracts (top panel) and Ltk- cells cotransfected with both hDAT and -synuclein (bottom panel). Middle panel denotes the blockade of neuronal DAT immunoprecipitation by -synuclein antibodies upon the addition of DAT-CT583–620-GST fusion protein (50 µg). c) DAT antibodies coimmunoprecipitated -synuclein from extracts of rat striatum (top panel) and cotransfected Ltk- cells (bottom panel). d) Western blot of rat striatal DAT after affinity purification with GST fusion protein encoding the NAC domain of synuclein, but not by GST amino- or carboxyl-terminal peptides of -synuclein or GST alone. e) Colocalization of hDAT and -synuclein in cultured progenitor dopaminergic cells. Confocal optical sections of the distribution of hDAT (top) and -synuclein (middle) in cultured neurons were obtained by double immunostaining with DAT monoclonal and -synuclein polyclonal antibodies. DAT proteins were visualized by TRITC-conjugated secondary antibodies (red) and -synuclein through FITC (green). Bottom panel: Superimposition of confocal images. Arrowheads highlight areas of punctate -synuclein and hDAT clustering at the cell surface.

The physical association of DAT and -synuclein was further confirmed by coimmunoprecipitation experiments. As illustrated in Fig. 1b , -synuclein antibodies precipitated a band of relative molecular mass of 83 kDa that immunoreacted with hDAT antibodies in extracts of both native striatal tissue (top panel) and Ltk^- cells transiently coexpressing the hDAT and -synuclein (bottom panel). Coimmunoprecipitation of the native striatal DAT by -synuclein antibodies was completely blocked by coincubation with GST fusion proteins encoding the DAT-CT^583–620 tail amino acids, but not by GST fusion proteins alone (Fig. 1b , middle panel) or those encoding the hDAT amino-terminal (data not shown), which suggests that -synuclein and DAT protein complex formation is dependent on direct binding to these DAT-CT tail sequence motifs in situ. Conversely, as depicted in Fig. 1c , hDAT receptor antibodies immunoprecipitated a band of apparent M_r 21,000 immunoreactive for -synuclein from both extracts of native striatal (top panel) and cotransfected Ltk^- cells (bottom panel), indicating the existence of physiologically relevant complex formation between these two presynaptic holoproteins in vivo.

To delineate the region and amino acid sequence motifs of -synuclein that contribute to the putative direct protein–protein binding domain with hDAT-CT tails, we incubated native rat striatal brain extracts with GST fusion proteins encoding various structural domains of the cloned human -synuclein protein (Fig. 1d ). Although control GST-alone did not bind striatal DAT, the core region of -synuclein [K⁵⁸-P¹⁰²] (-syn2) that encodes the NAC domain, containing amino acid residues of the non-Aß amyloid peptide fragment found in Alzheimer’s disease plaques (8 , 35 36 37 38) , recognized and specifically bound to sequences within DAT (Fig. 1d ). GST fusion proteins encoding several different regions of -synuclein, including the amino lipid binding domain [M¹-E⁵⁷] (-syn1) and the acidic CT tail [Q¹⁰⁹-A¹⁴⁰] (-syn3), all failed to bind to and precipitate neuronal DATs (Fig. 1d ). These data clearly suggest that the direct binding of amino acid sequence motifs within the distal portion of the CT tail of DAT with the NAC domain of -synuclein is both necessary and sufficient for the maintenance and formation of relevant striatal DAT--synuclein complexes in vivo and in vitro.

Colocalization of hDAT and -synuclein in neuronal culture
The direct protein–protein complex formation between hDAT and -synuclein described above requires that these two protein moieties be coexpressed within the same neuron. Although reports of the distribution profiles of DAT and -synuclein (39 40 41 42) seem to be consistent with this contention, direct evidence for DAT and -synuclein colocalization in dopaminergic neurons is lacking. Figure 1e depicts the immunoconfocal imaging and subcellular distribution pattern of -synuclein and DAT in cultured human substantia nigral neuronal precursor cells. The strong overlap in the clustering of hDAT and -synuclein in these neurons suggests that -synuclein, by virtue of its close spatial proximity to hDAT, may indeed subserve a distinct functional modality by preferentially modulating dopaminergic presynaptic function.

Functional characterization of the hDAT--synuclein interaction
The DAT is a major determinant of dopaminergic neurotransmission and synaptic strength since it is the primary mechanism by which endogenous neurotransmitter is removed rapidly from the synaptic cleft. Increases or decreases in DAT function will concomitantly decrease or increase synaptic DA concentrations, respectively, thereby regulating the activity of multiple post- and presynaptic dopamine D1 and D2 like receptor-mediated events (24 , 25 , 43) . The existence of neuronal hDAT--synuclein protein–protein binding complexes suggests that functionally relevant interactions occur between these two presynaptic protein moieties. We therefore assessed the activity of the hDAT in cells coexpressed with -synuclein. As illustrated in Fig. 2a , the translocation velocity of cellular DA uptake was significantly (P<0.01) increased in Ltk^- cells coexpressing human -synuclein relative to cells expressing hDAT alone. Thus, the estimated V_max for DAT-mediated [³H]DA uptake was enhanced by 50%, with no observable change in the estimated K_m of the hDAT for its preferred substrate in -synuclein coexpressing cells (Table 1 ). The apparent enhancement of cellular DA uptake was not due to an -synuclein-induced modification in the recognition of dopamine for the ligand binding domain of DAT nor was it a result of increased hDAT expression levels, since 1) as depicted in Fig. 2c , the estimated affinity of DA at the ligand binding site of hDAT in -synuclein coexpressing cells (K_i, 748±90 nM) was not significantly different from that observed for cells expressing hDAT alone (K_i 698±86 nM); and 2) as illustrated in Fig. 2d , the estimated Bmax or site density of whole cell hDATs as indexed by the saturable binding of [³H]CFT was not significantly different in control hDAT-expressing cells (145±12 fmol/10⁵cells) or in cells cotransfected with -synuclein (138±15 fmol/10⁵cells), findings consistent with data obtained by Western blots of hDAT (data not shown) and by double-label immunoconfocal microscopy of these cells (see Fig. 3 , below). The increase in hDAT-mediated [³H]DA uptake by -synuclein is not restricted to Ltk^- cells and is seen in both cotransfected COS-7 and HEK293 cells (see below).

View larger version (35K): [in this window] [in a new window]   Figure 2. -Synuclein-mediated enhancement of [3H]DA uptake. a) Representative saturation plots of [3H]DA uptake in Ltk- cells expressing the hDAT alone or coexpressing -synuclein revealed an increase in the Vmax for [3H]DA uptake accumulation (n=8, P<0.01) with no significant alteration in estimated Km values; listed in Table 1 (inset: linear transformation of the data). b) -Synuclein-mediated increase in [3H]DA uptake by hDAT was blocked with the coexpression of a minigene encoding the DAT-CT583–620 peptide but does not alter the uptake of cells expressing hDAT alone (n=3, P<0.01). c) Representative curves of the dose-dependent inhibition of [3H]2ß-carbomethoxy-3ß-(4-fluorophenyl)tropane (CFT) (4 nM) binding by dopamine to hDAT. Coexpression of -synuclein and hDAT did not alter the estimated affinity value of dopamine for hDAT (Ki values listed in the text). d) Estimated whole cell hDAT protein densities or Bmax values in control or -synuclein coexpressing cells. Values represent the mean ± SE of 4 independent experiments, each conducted in duplicate.

View this table: [in this window] [in a new window]   Table 1. Kinetics of [3H]DA uptake in Ltk- cells transiently expressing wild-type mutant hDAT alone or with -synucleina

View larger version (63K): [in this window] [in a new window]   Figure 3. -Synuclein-mediated membrane targeting of hDAT. Confocal microscopy of Ltk- cells expressing -synuclein, hDAT, or both were immunolabeled with DAT (1:1000) monoclonal antibodies and/or -synuclein (1:1000) polyclonal antibodies as described. DAT proteins were visualized by TRITC-conjugated secondary antibodies (red) and -synuclein through FITC (green), with colocalization depicted by yellow staining on the merged panels. When hDAT was expressed alone, immunolabeling was found to be dispersed throughout the cell (top panel) whereas cells coexpressing -synuclein and DAT revealed DAT immunoreactivity at the cell surface (middle panel), an effect completely abolished by the expression of the DAT-CT583–620 peptide (lower panel). No staining was observed when primary antibodies were eliminated. Data are representative of 4–8 independent experiments, with a total cell sampling of more than 1000.

Additional evidence for the participation of functional hDAT--synuclein protein–protein complexes in causing the selective increase in maximal hDAT-mediated DA uptake without altering ligand binding density or substrate affinities was obtained with the use of a minigene encoding sequences of the hDAT-CT tail. As described in Fig. 1b, we reasoned that in hDAT and -synuclein coexpressing cells, the additional expression of CT tail peptide sequence motifs would act as a competitive inhibitor of putative -synuclein-hDAT protein recognition domains. As depicted in Fig. 2b , the coexpression of the minigene encoding the peptide DAT-CT^583–620 severely inhibited the modulation of hDAT-mediated DA translocation by -synuclein. In control hDAT-expressing cells, the DAT-CT tail^583–620 peptide (see Fig. 2b ) did not modulate either the K_m or V_max of cellular [³H]DA uptake mediated by hDAT (30) .

To determine if the observed -synuclein-mediated enhancement of [³H]DA uptake by hDAT is the sole product of hDAT--synuclein protein–protein complex formation, we used hDAT transporter mutants in which the CT tail was truncated or swapped with amino acid sequence motifs encoded by the intracellular CT tail of totally unrelated proteins: that of dopamine D1 or D5 G-protein-coupled receptors. These mutants have already been characterized and display functional DA uptake characteristics and affinities for the endogenous neurotransmitter DA more reminiscent of native neuronal DATs (30) . As listed in Table 1 , cells coexpressing -synuclein and hDAT-CT tail truncation mutant (Glu⁵⁹⁸), which does not contain sequences encoding the putative binding domain with -synuclein, displayed DA uptake affinity and V_max characteristics virtually identical to those of cells expressing hDAT-CT-truncated mutants alone. Similarly, the swapping of the hDAT-CT tail with sequences encoded by the intracellular CT tail of dopamine D1 G-protein-linked receptors (hDAT-D1_CT^A332-T446), completely failed to reconstitute -synuclein-mediated increases in cellular [³H]DA translocation rates (Table 1) despite extreme differences in deduced amino acid sequence homology, net charge, and overall length. Identical results were obtained with hDAT mutants in which the CT tail was replaced by highly variable sequences encoded by another member of the D1 receptor family, the dopamine D5 receptor (hDAT-D5_CT^A360-H447; data not shown), indicating the specificity in -synuclein-hDAT-CT coupling. Taken together, these data indicate that the binding of DAT-CT tail^583–620 sequences to -synuclein is both sufficient and necessary for the expression of -synuclein-mediated functional regulation of the DA translocation process.

Coexpression of -synuclein enhances the recruitment of hDAT to the cell surface
Previous work has suggested that the primary mechanism whereby the uptake velocity of the presynaptic DAT is decreased is via the rapid internalization of cell surface DATs into various intracellular compartments (44 45 46) . The observed selective enhancement of [³H]DA uptake by -synuclein suggested to us of the possibility that -synuclein-mediated augmentation of hDAT function may be the result of the recruitment of constitutively internalized pools of hDAT to the plasma membrane. Figure 3 depicts the confocal images and colocalization patterns of -synuclein and hDAT when expressed in Ltk^- cells. Confocal immunofluorescent microscopy of Ltk^- cells expressing the human DAT or -synuclein alone (top panel) indicates that these proteins are expressed quite diffusely throughout the cell with significant intracellular and cell surface plasma membrane localizations. These observations are in line with the reported subcellular distribution patterns of DAT and -synuclein in both native neuronal and cultured cells (47 48 49) (also see Fig. 1e , above). On coexpression with -synuclein, however, the widespread intracellular distribution of hDATs is substantially diminished (Fig. 3 , middle panel), instead being manifested by hDAT immunoreactivity material located primarily on the cell surface. Moreover, in cell lines such as HEK293, where some studies report that a majority of hDATs are preferentially targeted to the cell surface with decreased immunoreactive material seen intracellularly, coexpression of -synuclein mediates the formation of large hDAT clusters on the cell surface (Fig. 4 ). In these cells, -synuclein similarly produces corresponding increases in functional [³H]DA uptake [K_m(µM): control hDAT: 4.5±0.53 vs. hDAT+-synuclein 5.1±0.47; V_max (pmol/10⁵cells/min): control hDAT 0.6±0.04 vs. hDAT+-synuclein 0.94±0.06, n=3, P<0.05].

View larger version (62K): [in this window] [in a new window]   Figure 4. Targeting of hDAT in HEK293 cells. Confocal microscopy of HEK293 cells expressing -synuclein, hDAT, or both were immunolabeled with DAT (1:1000) monoclonal antibodies and/or -synuclein (1:1000) polyclonal antibodies as described. DAT proteins were visualized by TRITC-conjugated secondary antibodies (red) and -synuclein through FITC (green), with colocalization depicted by yellow staining on the merged panel. hDAT expressed in HEK293, in the absence of -synuclein, exhibited dispersed immunolabeling throughout the cell (top panel). HEK293 cells coexpressing -synuclein and DAT revealed DAT localized at the cell surface with pronounced punctate immunolabeling, as indicated by arrows (lower panel). No staining was observed when primary antibodies were eliminated. Data are representative of 4 independent experiments with a total cell sampling of more than 1000.

The -synuclein-mediated recruitment or maintenance of hDATs on the cell surface appears to be the product of the direct protein–protein binding complex between these two protein moieties. As illustrated in Fig. 3 (bottom panel), coexpression of a minigene encoding sequences of the DAT-CT tail^583–620 completely prevents -synuclein-mediated recruitment of hDAT to the plasma membrane and displays a subcellular distribution pattern similar to that observed in cells expressing hDAT alone. Virtually identical results were obtained when cells were cotransfected with hDAT-D1_CT or D5_CT mutants (data not shown). Only minor changes in the distribution pattern of -synuclein were noted under these conditions, suggesting that -synuclein-hDAT complex formation is transient, reversible, and dynamically regulated. Pretreatment of cells with hDAT transporter substrates such as DA or amphetamine (10 µM), however, did not alter -synuclein-mediated enhancement of either hDAT function or subcellular distribution profiles, which suggests that, at least under these conditions, -synuclein-DAT complex formation is regulated by means other than those associated with substrate translocation. In any event, these data suggest that -synuclein-mediated enhancement in the functional uptake of [³H]DA by hDAT is achieved by diminishing the proportion of hDATs found in intracellular compartments, increasing the number of functionally relevant transporters available at the cell surface. Moreover, these events appear to be the sole product of the direct binding of the hDAT-CT tail to sequence motifs of -synuclein in a substrate-independent fashion. Whether -synuclein targets or tags internalized pools of hDATs into cell surface ‘recycling’ pathways, functionally rescues transporters from endosomal/lysosomal degradative fates (44 45 46) , or both, is unknown.

DA-induced apoptosis in transfected cells
Previous in vitro studies have shown that DA can induce apoptosis in both neuronal and non-neuronal cell cultures (50) . The selective increase in hDAT-mediated [³H]DA uptake by -synuclein, as the result of transporters recruited to the cell surface, provided the potential for enhanced cellular apoptosis upon exposure to DA. To examine this possibility, both transfected and untransfected HEK293 cells were incubated with 50 µM DA for 5 h and apoptosis was detected using the ApoAlert MitoSensor kit (Clontech). In untransfected cells, only a small proportion of cells exhibited apoptosis for both control and DA-treated cells control: 4.4% apoptotic (DA-treated: 7.3% apoptotic, n=3, P<0.01) whereas DA-induced cellular apoptosis, as determined through fluorescence microscopy, in hDAT-expressing cells was slightly greater than untreated cells control: 7.1% apoptotic (DA-treated: 10% apoptotic, n=3, P<0.01; Fig. 5 , top panel). As depicted in Fig. 5 (middle panel), there was a significant increase (+20%, P<0.05. n=3) in DA-induced apoptosis in cells coexpressing both hDAT and -synuclein control: 8% apoptotic (DA-treated: 31% apoptotic, n=3, P<0.01) compared with hDAT alone. This enhancement of DA-induced apoptosis by -synuclein was blocked by the coexpression of the DAT-CT tail^583–620 minigene control: 8.8% apoptotic (DA-treated: 13'% apoptotic, n=3, P<0.01; Fig. 5 , lower panel). Whereas the DAT-CT tail^583–620 minigene has no apparent functional effect on hDAT itself with respect to both uptake capacity and affinity (Fig. 2b ) (30) , the ability of the minigene to block both the enhancement of DA uptake and apoptosis in cells coexpressing both hDAT and -synuclein can be attributed to the disruption of the hDAT--synuclein protein–protein interaction by the competitive inhibition of the DAT CT tail peptide. These data clearly illustrate that the hDAT--synuclein protein–protein interaction not only enhances transporter uptake velocity as the result of an increased population of DAT found on the cell surface, but can also lead to subsequent acceleration of DA-induced apoptosis.

View larger version (134K): [in this window] [in a new window]   Figure 5. Detection of DA-induced apoptotic cells. Immunofluorescence imaging of HEK293 cells expressing either hDAT alone, both hDAT and -synuclein, or hDAT and -synuclein with the DAT-CT583–620 peptide after treatment with an apoptosis detection reagent (ApoAlert MitoSensor, Clontech). Healthy cells display punctate red fluorescence with the accumulation of the ApoAlert MitoSensor reagent in mitochondria whereas apoptotic cells fluoresce green, with the reagent remaining in the cytoplasm. Although untreated cells show largely red fluorescence, cells incubated with 50 µM DA for 5 h show a slight increase in the population of apoptotic cells, as seen with cells expressing hDAT alone (top panel). Cells coexpressing both hDAT and -synuclein show further significant increases in DA-induced apoptosis (middle panel), which is abolished with the expression of DAT-CT583 minigene (lower panel). Untransfected HEK293 cells exhibit only modest levels of DA-induced apoptosis (data not shown). Data are representative of 3 independent experiments with a total cell sampling of more than 4500.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The crucial importance ascribed to presynaptic DAT function in the control of the synaptic availability of DA suggests that its regulation may be dynamically controlled for the maintenance of normal dopaminergic neurotransmission. We describe here the first identified direct binding partner for the DAT and suggest a previously unappreciated functional role for neuronal -synuclein by which the activity of the DAT and subsequently dopaminergic neurotransmission may be modulated. The direct protein–protein interaction between hDAT and -synuclein was found to involve the hDAT-CT tail, specifically the last 15 amino acids of the hDAT (Glu⁵⁹⁸-Val⁶²⁰) and the NAC domain of -synuclein. The identification of the direct interaction between these two proteins has ascribed novel roles for both the hDAT-CT tail and the NAC domain of -synuclein.

The NAC region was first identified as a peptide associated with ß-amyloid protein in extracellular amyloid plaques found in the brains of patients with Alzheimer’s disease. A few studies have implicated the NAC peptide to be involved in fibrillation (51 52 53) and perhaps cytotoxicity (53 54 55) ; nevertheless, relatively little is known about the role of the NAC domain. Earlier we had shown the hDAT-CT tail to play an important role in the function of the DAT (30) , but our present study has identified an additional function for hDAT-CT tail. This protein–protein interaction not only increased DAT-mediated DA uptake but moreover, appears to be correlated to a preponderance of DAT on the cell surface, compared to cells expressing hDAT only. Previous studies have implicated DAT trafficking as a regulatory mechanism. The DAT has been shown to exhibit significantly reduced DA uptake capacity on PKC-stimulation (44 , 56 57 58) . This PKC-mediated reduction of DAT activity was correlated with a decrease in DAT localization on the cell surface (44) . Furthermore, the trafficking mechanism by which the DAT is regulated has been more clearly elucidated, identifying and involving either the recycling (45) or lysosomal pathways (46) . Evidence in this study, including the enhanced DAT function when coexpressed with -synuclein and the inability of -synuclein to affect mutant DATs with respect to both the intracellular and cell surface population of the mutant DATs, would lead us to hypothesize that the up-regulation in DAT activity can be attributed to the increased trafficking of DATs to the cell surface. Although this is the first identified direct binding partner for the DAT, the ability of ‘accessory’ binding proteins to regulate transporter is not unprecedented. Other distantly related transporters have been shown to be associated with accessory proteins, including the GABA transporter (GAT1) with syntaxin-1A (59) and the glucose transporter (GLUT1) with calnexin (60) and GLUT1CBP (61) .

We propose that by the direct binding of sequences of the hDAT-CT tail to the NAC domain of -synuclein, a functionally relevant complex forms between these two proteins in both expressed cells and striatal neurons that allows for the effective targeting of DAT proteins to the cell surface, thereby increasing functional DA uptake. Moreover, -synuclein-deficient transgenic mice exhibited an attenuated locomotor response to amphetamine (22) . Since amphetamine is known to exert its psychostimulant effects through the DAT, it is possible that with -synuclein-deficient mice, attenuation of the amphetamine response may be due to the inability of the DAT to be up-regulated by -synuclein. These data, then, support a potential role for -synuclein in regulating normative-dopaminergic tone and an involvement with the initial and adaptive responses of dopaminergic neurons to various drugs of abuse, such as cocaine and amphetamine, that block DAT activity and, in the expression of aberrant DAT, function in dopaminergic neuropsychiatric disease states such as attention deficit disorder and schizophrenia (62 63 64 65) .

Because -synuclein is expressed in many regions of the brain and is toxic to numerous cells when overexpressed, the exact role, if any, -synuclein may play in mediating the observed selective degeneration of substantia nigral dopaminergic neurons in Parkinson’s disease is enigmatic (66 67 68) . In addition to the enhanced DA uptake, coexpression of -synuclein and hDAT accelerates DA-induced apoptosis in HEK293 cells. This acceleration in apoptosis appears to be correlated to the increase in DA uptake levels in cells coexpressing the hDAT and -synuclein mediated by the increased population of DAT on the cell surface. DA-induced apoptosis has been documented in both recombinant and neuronal cells (50 , 69 , 70) . Intracellular DA can be metabolized into reactive oxygen species (ROS) that are capable of inducing apoptosis through mitochondrial alterations, which leads to the release of cytochrome c (71 72 73) through activation of the JNK pathway (50 , 73) or by the activation of AP-1 and NF-B transcription factors (74) . The attenuation of apoptosis induced by DA or DA-like molecules by either antioxidants (75 , 76) or the expression of proto-oncogene bcl-2, whose anti-apoptotic effects have been attributed to the reduction of ROS production (77) , provides corroborating evidence in the role of oxidative metabolism in DA-induced apoptosis. Although the neurotoxic effects of -synuclein aggregates provide evidence for the direct involvement of -synuclein in cell death (6 , 53 54 55) , along with the high expression of -synuclein in degenerated neurons of MPTP-treated baboons (78) , it appears that -synuclein-mediated cell death does not include the classical apoptotic pathway (79 , 80) . Moreover, there is evidence that -synuclein fibrillation, characteristically seen in Lewy bodies, may require the presence of oxygen free radicals (6 , 32 , 81) . One may speculate that the aggregation of -synuclein in Lewy bodies, which may promote the degeneration of dopaminergic neurons in PD, could be the product of apoptotic events.

The fact that hDAT and -synuclein form functional protein–protein complexes in the regulation of DAT function, which we have shown leads to enhanced DA-induced apoptosis, may provide a heuristic framework in which to view selective dopaminergic cell death in PD and provide potential therapeutic strategies to combat this disease state. This contention is particularly intriguing, since in addition to -synuclein, the functional activity of the DAT itself has been implicated in the etiology of PD. Thus, the selective vulnerability and destruction of dopaminergic neurons in Parkinson’s disease has been ascribed to the uptake of dopamine or possibly other neurotoxins by the DAT and that the subsequent intracellular metabolism of these compounds leads to oxidative stress, mitochondrial damage and cell death (4 , 5 , 50 , 82 83 84) . Therefore, the capability of these compounds to promote dopaminergic neurodegeneration and parkinsonian-like movement disorders is dependent on the expression and activity of the DAT (29 , 85) . Perhaps because of disease-induced mutation or over/underexpression of -synuclein in dopamine neurons, aberrant complex formation between -synuclein and DAT may increase the cellular accumulation of DA or some other metabolite to levels that initiate a chain of events leading to oxidative stress and specific dopaminergic cell death. Since oxidative stress further increases -synuclein aggregation (32 , 81) , additional work in this area appears warranted.

	ACKNOWLEDGMENTS

 
The authors wish to thank Brian Vukusic for excellent technical assistance and F. Ko and N. Zhu for help with initial experiments. This work was supported in part by a grant from the Natl. Parkinson’s Foundation and the MRC of Canada (to H.B.N.). F.J.S.L. is a recipient of an OMHF Studentship, F.L. is a fellow of the CPRF, and Z.B.P. is a NARSAD Young Investigator. In memory of Dr. H. B. Niznik.

Received for publication June 7, 2000. Revision received October 16, 2000.

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