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Dopamine and Cu1/21 Can InduceOligomerization of a-Synuclein in theAbsence of Oxygen: Two Types ofOligomerization Mechanisms for a-Synucleinand Related Cell Toxicity Studies
Yonghwang Ha,1,2 Aerin Yang,1 Seyoung Lee,1 Kibong Kim,1 Hyunjeong Liew,3
Sang Hyung Lee,4 Ju Eun Lee,5 Hong-In Lee,5 Yoo-Hun Suh,6,7*Hee-Sung Park,1* and David G. Churchill1*1Department of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), Daejeon,Republic of Korea2Convergence Science and Technology Research Center (CSTRC), Jungwon University, Chungbuk,Republic of Korea3Convergent Research Consortium for Immunologic Disease I Cardiovascular Research Laboratory,Annex to Seoul St. Mary’s Hospital, Seoul, Korea4Department of Neurosurgery, SMG-SNU Boramae Medical Center, College of Medicine, Seoul NationalUniversity, Seoul, Korea5Department of Chemistry and Green-Nano Materials Research Center, Kyungpook National University,Daegu, Republic of Korea6Department of Pharmacology, College of Medicine, Seoul National University, Seoul, Korea7Korea Brain Research Institute, Daegu, Republic of Korea
a-Synuclein oligomers can induce neurotoxicity and areimplicated in Parkinson’s disease etiology and diseaseprogression. Many studies have reported a-synuclein oli-gomerization by dopamine (DA) and transition metal ions,but few studies provide insight into joint influences of DAand Cu21. In this study, DA and Cu21 were coadminis-tered aerobically to measure a-synuclein oligomerizationunder these conditions. In the presence of oxygen, DAinduced a-synuclein oligomerization in a dose-dependentmanner. Cu1/21 did not effect oligomerization in such a
manner in the presence of DA. By electrophoresis, Cu21
was found easily to induce oligomerization with DA. Thisimplies that oligomerization invoked by DA is reversible inthe presence of Cu21, which appears to be mediated bynoncovalent bond interactions. In the absence of oxygen,DA induced less oligomerization of a-synuclein, whereasDA/Cu21 induced aerobic-level amounts of oligomers,suggesting that DA/Cu21 induces oligomerization inde-pendent of oxygen concentration. Radical species weredetected through electron paramagnetic resonance (EPR)
Additional Supporting Information may be found in the online version of
this article.
Y. Ha and A. Yang contributed equally to this work.
Contract grant sponsor: National Research Foundation (NRF) of Korea;
Contract grant numbers: 2011-0017280 (to D.G.C.); Contract grant
number: 2010-0004891 (to H.-S.P.); Contract grant sponsor: Advanced
Biomass R&D Center; Contract grant number: 2011-0031357 (to
H.-S.P.); Contract grant sponsor: Establishment and Operation of Korea
Brain Research Institute (KBRI) Basic Research Program of the Ministry
of Science, Ict & future Planning; Contract grant number: 2031-415 (to
Y.-H.S.); Contract grant sponsor: Brain Research Center of the “21st
Century Frontier Research Program,” funded by the Ministry of Educa-
tion, Science and Technology, of the Republic of Korea; Contract grant
number: 2011k000270 (to Y.-H.S.); Contract grant sponsor: Converging
Research Center Program funded by the Ministry of Education, Science
and Technology; Contract grant number: 2011k000566 (to Y.-H.S.);
Contract grant sponsor: Basic Science Research Program through the
NRF of Korea funded by the Ministry of Education, Science and Tech-
nology, Contract grant number: 2011-0027273 (to H.-I.L.).
*Correspondence to: David G. Churchill, Department of Chemistry,
Korea Advanced Institute of Science and Technology (KAIST), 373-1
Guseong-dong, Yuseong-gu, Daejeon, 305-701 Republic of Korea.
E-mail: [email protected] or Hee-Sung Park, Department of Chem-
istry, Korea Advanced Institute of Science and Technology (KAIST),
373-1, Guseong-dong, Yuseong-gu, Daejeon, 305-701 Republic of
Korea. E-mail:[email protected] or Yoo-Hun Suh, Department of Phar-
macology, College of Medicine, Seoul National University, Seoul, Korea,
Korea Brain Research Institute, Daegu, Republic of Korea.
E-mail:[email protected]
Received 5 August 2013; Revised 30 September 2013; Accepted 14
October 2013
Published online 29 November 2013 in Wiley Online Library
(wileyonlinelibrary.com). DOI: 10.1002/jnr.23323
VC 2013 Wiley Periodicals, Inc.
Journal of Neuroscience Research 92:359–368 (2014)
spectroscopic analysis arising from coincubation of DA/Cu21 with a-synuclein. Redox reactions induced by DA/Cu21 were observed in multimer regions of a-synucleinoligomers through NBT assay. Cellular toxicity resultsconfirm that, for normal and hypoxic conditions, copperor DA/Cu21 can induce cell death, which may arise fromcopper redox chemistry. From these results, we proposethat DA and DA/Cu21 induce different mechanisms of a-synuclein oligomerization, cross-linking with noncovalent(or reversible covalent) bonding vs. likely radical-mediated covalent modification. VC 2013 Wiley Periodi-cals, Inc.
Key words: a-synuclein; dopamine; copper; proteincross-linking; radical-mediated chemistry
Among the various neurodegenerative diseases,Parkinson’s disease (PD) represents a major debilitatingillness; molecular aspects of this principal disease affect-ing motor function has not been well elucidated, spur-ring ongoing research. One of the main aspects of PD,separating it from Alzheimer’s disease (AD), is the areaof the brain affected and the principle neurochemicals inplay. Dopaminergic neurons in the substantia nigra (SN)are affected, so in the human brain a decrease of dopa-mine (DA) concentration occurs (Uversky, 2007). Tocompensate for DA, L-DOPA is used in the treatmentof alleviation of symptoms of PD, e.g., bradykinesia,rigidity, and resting tremor (Kostrzewa et al., 2002). DAis a very reactive catechol/primary amine (Rees et al.,2009) and plays a very important neurological role as aneurotransmitter and is also a precursor to epinephrine/norepinephrine, central neurotransmitters in brain neu-ron chemistry (Lee et al., 1999; Burke et al., 2004). Itcan be easily oxidized to quinine and indole and“polymerized” to neuromelanin (Bisaglia et al., 2007).This diverse reactivity allows for great complexity in themolecular aspects of this disease. Although manyresearchers have tried to rationalize the observeddecrease of DA, the precise reason for this loss is not asyet unknown.
Lewy body deposition from midbrain and neocorti-cal regions is another main characteristic in moreadvanced stages of PD (Braak et al., 2003). It has beenestablished that the main component of Lewy bodydeposits is the protein a-synuclein (a-Syn). a-Syn iscommonly phosphorylated at positions 87 or 129 or both(Spillantini et al., 1997). Lewy bodies and related growthsare a deposit of fibril formation in a-Syn. Many studieshave shown the formation of a-Syn fibril is related to anincreasing pleated sheet structure. Transition metal ionsor low-MW aldehydes, e.g., 4-hydroxynonenal (Qinet al., 2007) or 4-oxononenal (Nasstrom et al., 2009), canallow for the oligomer formation of a-Syn. Such aggrega-tion seems to be a general trend with various neurodege-nerative disease-related proteins and various other diseasesas well. It is now understood that oligomers of a-Syn aremore toxic than the fibril form (Brown, 2010; Pountneyet al., 2005; Wright et al., 2009).
Copper is one of the most important redox activetransition metal ions in biology. It is found in the activesite of biological enzymes for oxygenation/deoxygena-tion in cellular metabolism (Solomon et al., 2001). Asdescribed elsewhere, copper ions are controlled strictlyin neuronal cells; e.g., uptake, transfer, distribution, andregulation of metabolism are all highly regulated (Koz-lowski et al., 2009). However, the aging or toxic mate-rial can induce the release of free copper ions, whichallows for interactions with a-Syn and DA. Many stud-ies have brought out that copper binds very favorably toprotein donor analytes and, in so doing, induces struc-tural changes. Copper can bind by alternate modes tovarying degrees: N-terminal region (strong) or C-terminal region (weak; Rasia et al., 2005). The ion canalso change the structure of a-Syn from random into a-helix in membranes (Lucas and Lee, 2011) and mediateredox reaction in the presence or absence of oxygen(Paik et al., 2000; Lucas et al., 2010; Wang et al.,2010). Finally, it was found that Cu(II) can induce a-Syn oligomer in the presence of H2O2 (Paik et al.,2000).
From recent reports, it is clear that PD is closelyrelated to DA, a-Syn, and transportable transition metal ionsof abundance in the human brain. In regions of the synapseof neurons, their positions are close enough that they caninteract with one another (Lotharius and Brundin, 2002;Kozlowski et al., 2009). Under normal conditions, thesecomponents are controlled strictly. When small amounts ofCu are released, aggregation of a-Syn can be induced.
Many studies have shown the relation between twofactors, e.g., transition metal ions and synuclein (36 hits inISI Web of Science by January, 2013) or DA and synu-clein (135 hits in ISI Web of Science in January, 2013).However, few studies showed transition metal ions, DAand a-Syn in concert. Here we focus on the interplayamong a-Syn, copper ions, and DA together in vitro. Weexpected that in vitro study will help to show the rela-tionships among them in the context of chemistry rele-vant to PD. Experiments were performed to acquireinformation about how copper and DA can induce theoligomer form of a-Syn in relation to (an)aerobicconditions.
MATERIALS AND METHODS
General Comments About Protein Assays andOligomer Formation
a-Syn, DA, Cu1, and Cu21 were coincubated at variousconcentrations under aerobic or anaerobic conditions. SDS-PAGE assays (Bio-Rad, Hercules, CA; a Mini-Protean tetra sys-tem) were performed to measure aggregation of a-Syn underdifferent incubation conditions. Sample absorbances were meas-ured in obtaining binding information among protein, DA, andcopper ions. A nitroblue tetrazolium (NBT) assay (Paz et al.,1991; Akagawa et al., 2006) and electron paramagnetic resonance(EPR) analysis were used to verify whether paramagnetic orredox species were generated during the reaction.
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Construction of Plasmids for Synthesis of a-Syn
pCDF Duet-a-syn-wt was created by adding a codon-optimized a-Syn gene into pCDFDuet-1 using BamHI andAscI sites. Before the addition of the gene into the vector, aTEV protease site (ENLYFQG) was added N-terminally to thea-Syn gene through the use of the polymerase chain reaction(PCR). The resultant plasmid was constructed to express therecombinant protein His6-TEV protease site-a-syn-wt. Plas-mids were prepared by the method of Park and colleagues(Park et al., 2011; Lee et al., 2013).
Protein Expression and Purification
Plasmid pCDFDuet-a-Syn was transfected into Esche-richia coli BL21(DE3). A single colony was used to inoculate 10ml of the LB medium, supplied with 50 lg/ml kanamycin, 10lg/ml tetracycline, and 50 lg/ml streptomycin. This preculturewas incubated overnight at 30�C. Ten milliliters of the precul-ture was used to inoculate 400 ml of the LB medium. The cul-ture was grown at 30�C. When OD 600 nm reached 0.6,protein expression was induced by adding 0.5 mM IPTG for 19hr at 30�C. The cells were harvested by centrifugation andstored at 270�C.
The cell pellet was resuspended in lysis buffer (50 mMTris-HCl, pH 8.0, 300 mM NaCl, 14.3 mM 2-mercaptoethanol, 1 mg/ml lysozyme, 10 mM imidazole, prote-ase inhibitor cocktail; completely EDTA-free) and incubatedon ice for 20 min before being subjected to sonication. The celllysate was centrifuged at 10,000 rpm for 20 min; the superna-tant layer was then filtered through a 0.45-lm syringe filterprior to purification using a Ni-NTA resin (Bioprogen). After2 hr of incubation with Ni-NTA resin, the resin was washedwith 30 ml washing buffer (50 mM Tris-HCl, pH 8.0, 300mM NaCl, 14.3 mM 2-mercaptoethanol, 40 mM imidazole).Then, proteins were eluted in 0.8 ml of elution buffer (50 mMTris-HCl, pH 8.0, 300 mM NaCl, 14.3 mM 2-mercaptoethanol, 250 mM imidazole).
A portion of eluted protein solution (0.8 ml) was placedinto a 3500 MWCO dialysis bag and stirred through severalchanges of 500 ml 50 mM Tris-HCl buffer, pH 8.0, at 4�C.After dialysis, TEV protease buffer was added (203) to thesolution to make a 13 TEV protease buffer (50 mM Tris-HCl,pH 8.0, 0.5 mM EDTA, 1 mM dithiothreitol); 10 lg TEVprotease was then added to the solution. Proteins were digestedwith TEV protease at 37�C for 7 hr. After digestion was fin-ished, the protein solution was again dialyzed in the same way.Then, the protein solution was applied to Ni-NTA resin againto remove undigested protein and TEV protease. After 2 hr ofincubation with Ni-NTA resin, the supernatant layer was col-lected to obtain His-tag-free a-Syn.
Incubation of Protein With DA and Cu1/21
and SDS-PAGE
a-Syn was incubated with DA and various concentrationsof compounds at 37�C for 16–24 hr. To achieve anaerobicconditions, a glove box/dry box (Korea Kiyon company KK-01-AS model) was used. Ten millimolar phosphate buffer waspurged with nitrogen gas (99.51% purity) or argon (99.51%
purity) for more than 20 min. During insertion of samples andplacement of equipment into the glove box, the vacuum wasdrawn and purged (argon) for 10 min three times. After prepa-ration, each sample was sealed in a 1.5-ml tube and was incu-bated at 37�C for 16–24 hr. After incubation, SDS-PAGE andimmunoblotting were performed as previously described (Haet al., 2011).
Intensity Calculations From SDS-PAGE Data
For the analysis of intensity, ImageJ was used witholigomers; residual monomer was excluded.
NBT Assay
The NBT assay was applied to nitrocellulose paper afterSDS-PAGE and Western blotting. A 0.24 mM NBT solution(Sigma Aldrich, St. Louis, MO) was made by dissolution with 2.0M potassium glycerate solution, pH 10. Nitrocellulose paper afterWestern blotting was soaked with NBT solution and incubated ina dark room at room temperature for 40 min. Photos were takendirectly under wet condition of nitrocellulose-stained paper.
Absorbance
Sample absorbance was checked with a Nanodrop 2000instrument (Thermo Fisher Scientific, Fair Lawn, NJ) at roomtemperature. The amount of protein in the sample was 2 ll. Thewavelength determinations were made from 250 to 350 nm.
EPR
Samples for EPR experiments were prepared by incubatinga-Syn with DA with and without CuCl2 at 14�C in 10 mMsodium phosphate buffer at pH 7.4. Each sample was mixed withan equal volume of glycerol before being frozen in liquid nitro-gen in preparation for EPR measurement. Final concentrations ofa-Syn, DA, and Cu21 were 30 lM, 150 lM, and 150 lM,respectively. X-band (9 GHz) EPR spectra were collected on aJeol (Japan) JES-TE300 ESR spectrometer using a 100-kHz fieldmodulation. Low-temperature spectra were obtained using a JeolES-DVT3 variable temperature controller. EPR experimentalconditions are microwave (MW) frequency, 9.135 6 0.005 GHz;modulation amplitude, 1 G; time constant, 0.3 sec; scan speed,1,250/min; MW power 5.0 mW; and temperature, 110 K. Thespectral simulations were performed in the program EasySpin.
Cellular Toxicity Assay (WST-1)
Cellular toxicity assay was performed via the WST-1 col-orimetric assay (Roche Molecular Biochemicals, Mannheim,Germany) under aerobic and hypoxic conditions. For thehypoxic condition, the culture media and cultivation bottlewere purged for at least 30 min with Ar (99.991% purity). Thehuman neuroblastoma SH-SY5Y stable cell line was obtainedfrom ATCC (Manassas, VA). Cells were maintained in DMEMsupplemented with 20 mM glucose and 38 mM sodium bicar-bonate. The mixture was treated with 10% fetal bovine serum(Nalgene Gibco, Grand Island, NY). Cells were plated at 1 3104 cells/well (96-well plate) and were incubated with oligom-ers over 24 hr. After gentle agitation of the solution, assayswere performed according to manufacturer’s instructions. Next,spectrophotometric absorbances were measured; determinations
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at 450 nm were made using a microplate ELISA reader (Molec-ular Devices, Sunnyvale, CA). The absorbance of control cells,which were cells lysed with Triton X-100 (resting cells), wasset as 100%. These values were normalized relative to the con-trol group. Each sample was treated in triplicate.
RESULTS
Oligomerization of a-Syn by DA or DA/Cu21 inthe Presence of Oxygen
The characteristics of a-Syn oligomerization werestudied under various conditions in vitro with DA. Highlevels of aggregation could be observed when the ratio ofDA was increased, stoichiometries of 0, 1, 10, and 100(Fig. 1). Standard deviations (SDs) of aggregation werehigh at the ratio of 10 and 100. The mean value at theratio of 100 was less than that for the ratio of 10. Thismay be from high induction of aggregation by DA at
high concentration, which is over the detection range ofelectrophoresis, 260 kDa.
Interestingly, the dimer and trimer band positionscorrelate with increasing DA. When the concentration ofDA was high, the positions of dimer and trimer becamehigher, which implies increasing the molecular weight ofthese oligomers.
Dark precipitate formed in the incubation solutions(Supp. Info. Fig. 1). DA can be easily transformed intononhomogeneous polymeric forms in the presence ofoxygen, into DA-quinone, into DA-chrome, and eventu-ally into neuromelanin (Bisaglia et al., 2007). Naturalneuromelanin in the human brain also contains variousamino acids (e.g., Cys) or proteins, but a-Syn can also beincorporated, entrapped in neuromelanin granules locatedin the substantia nigra, characteristic in PD (Fasano et al.,2003).
From Figure 1, DA changes to, e.g., DA-neuromelanin in the presence of dioxygen and induces theaggregation of a-Syn in a dose-dependent manner.Oligomers induced by DA or DA derivatives were resistantto sodium dodecyl sulfate (SDS), which is consistent withother studies (Cappai et al., 2005; Leong et al., 2009).
Next, Cu1/21 ions were cotreated with a-Syn andDA (ratio 1: 1; Fig. 2). Although mean values showed asmall increase, SDs were very high, which suggests thatCu1/21 did not effect the aggregation of a-Syn coincubatedwith DA. There were no significant differences betweenCu1 and Cu21, which suggests that the conversion betweenCu1 and Cu21 under these conditions is facile.
Interestingly, copper gave a trend different fromthat for the DA-only assay, especially for dimers andtrimers. Lane 1 with DA and Syn (1:1) was set as thecontrol. When copper concentration increased, a lowerposition of dimer and trimer could be observed, relativeto the control, suggesting that copper can influence theoligomer induced by a-Syn and DA. This decreasedposition of dimer and trimer may be induced by a
Fig. 1. Left: SDS-PAGE of oligomerization of a-Syn (1 equiv., 7 lM)by DA incubation condition (various concentrations). Right: Calcu-lated relative intensities of oligomers except monomer. Experimentswere repeated three times (left shows one among three). Error bars sig-nify standard deviations. Incubation conditions were 37�C, 16 hr,sodium phosphate buffer (10 mM, pH 7.4). [Color figure can be viewedin the online issue, which is available at wileyonlinelibrary.com.]
Fig. 2. Effect of Cu1/21 on a-Syn oligomerization. SDS-PAGE results (a) and relative intensityand incubation ratio of a-Syn (b; 1 equiv., 7 lM), DA, and copper ions. Incubation conditionswere 37�C, 16 hr, sodium phosphate buffer (10 mM, pH 7.4). Experiments were repeated threetimes (a shows one among three experiments). Error bars indicate standard deviation values.
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complex of DA and Cu ion verified by the incubation ofDA with a-Syn first and addition of Cu21 after someperiod of time.
The band position of the Syn oligomer induced byDA or DA/Cu21 relative to Cu21/H2O2 is well knownin H2O2-induced Cu21 redox chemistry (Fig. 3). Theband induced by Cu21/H2O2 was the same as for DA/Cu21, which implies that the reaction by DA/Cu21 isrelated to redox reaction and DA did not attach a-Synduring aggregation. Therefore, a different mechanismmay exist in the presence of copper, which may be similarto H2O2/Cu21.
Next, another experiment was performed to deter-mine the effect of Cu on the aggregation of a-Syn:DA orCu21 added at different times. In lane 4 in Figure 3b,when copper was added 6 hr after DA addition, the posi-tions of dimers and trimers were the same as in lane 3. Inlane 5 of Figure 3b, when Cu21 was added first and DAwas added after 6 hr, the result was the same as that forlane 3. By comparing lanes 3, 4, and 5, the oligomeriza-tion induced by DA can be modulated by addition ofCu21, which suggests that the linkage of a-Syn inducedby DA is modifiable. Importantly, aggregation of a-Synby DA only is not irreversible, which suggests that DA isnot covalently attached to Syn. This result suggests thatthere are either no covalent bonds between DA and a-Syn, compatible with the result by Leong et al. (2009), orthat there are reversible covalent bonds at play. Oligomersinduced by DA are resistant to SDS (Cappai et al., 2005).Additionally, Rekas et al. (2010) suggested that DA-neuromelanin (DA polymer) may bind strongly with a-Syn, known to be SDS resistant. They suggested that
lysine residues of a-Syn may be involved in DA-melanincross-linking. Copper ions might induce the reverse reac-tion from DA-induced oligomerization as a result of metalion catechol binding and can mediate redox reactions(Sigel et al., 2006). Cu21 binding with DA catecholgroups may interrupt cross-linking. Conway et al. (2011)reported 14 molecules having catechol moieties thatinhibit fibrillation and stabilize protofibrils of a-Syn.Rees et al. (2009) also underscored the importance of cat-echols in DA or DA analogues for cross-linking. We pro-pose that Cu21 is catechol-bound in which the DA is inturn attached to a-Syn oligomers. This may function inblocking the cross-linking and induce reversibility andfinally may help reduce different types of aggregation.
Oligomerization of a-Syn by DA or DA/Cu21 inthe Absence of Oxygen
In obtaining information on the effects of oxygenon aggregation of a-Syn, experiments under anaerobicconditions were performed. In the absence of dissolvedoxygen (DO), no dark precipitate was detected in theincubation solution, even after 72 hr (data not shown),suggesting inhibition of neuromelanin production. Asshown in Figure 4, the positions of dimer and trimerbands induced by DA/Cu1/21 were almost the same asin lane 2 for Cu21. However, there was a small differencein the Cu1 assay. These results suggest that more attach-ment of DA to a-Syn and reverse reaction by Cu1 occursthan in the case of Cu21.
DA showed less protein oligomerization comparedwith results for aerobic conditions trials (�1.5-fold in lane2 relative to lane 1 in Fig. 4; an �4-fold increase in lane 2relative to the lane 1 in Fig. 1). This result indicates thatoxygen is an important factor for DA-induced oligomeri-zation. However, in the presence of DA/Cu1/21, a-Synoligomerization recovered to aerobic levels. In theabsence of oxygen, it is difficult for DA to be convertedinto neuromelanin. Therefore, we think that DA/Cu21
induces a mechanism different from that found in DA-neuromelanin cross-linking. Lucas et al. (2010) reportedthat Cu21 can be transformed into Cu1 in the absence ofDO, as determined via Cu K-edge X-ray absorption(XAS) spectral analysis. In this case, DA is present as amonomer in solution. Cu1 under redox conditions mayreact with DA and induce oligomerization in the absenceof DO. In the presence of dioxygen, the electron acceptorbetween Cu21 and DA is dioxygen. However, in theabsence of oxygen, the electron acceptor may be DA.The redox potential of DA is 0.174 (vs. Ag/AgCl), Syn-Cu21/Cu1 is 0.018 V (vs. Ag/AgCl; Wang et al., 2010),and Cu21/Cu1 is 20.036 V (vs. Ag/AgCl). During theredox reaction, the radical of DA can form. When theabsorbance of DA 1 Cu21, DA 1 a-Syn, and DA 1Cu21 1 a-Syn were checked, absorbance of DA 1 Cu21
showed spectra similar to DA 1 Cu21 1 a-Syn (Supp.Info. Fig. 2). This suggests that the spectrum of DA 1Cu21 1 a-Syn is induced mainly from DA 1 Cu21,
Fig. 3. a,b: The positional differences of dimer and trimer upon addi-tion of copper ions with various combinations of DA, Cu21, andH2O2. Lane 1: a-Syn (1 equiv., 7 lM); lane 2: a-Syn:DA (1:1); lane3: a-Syn:DA:Cu21 (1:1:10); lane 4: Cu21 addition (10 equiv.) after 6hr into the solution of a-Syn 1 DA (1:1); lane 5: DA (1 equiv.) addi-tion after 6 hr into the solution of a-Syn 1 Cu21 (1:10). Incubationconditions; sodium phosphate buffer (10 mM, pH 7.4), 37�C, 16 hr.
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which implies that Cu21 may be attracted more stronglyto DA than to a-Syn under the given conditions.
EPR and NBT Assay for Verification of RadicalSpecies by DA/Cu21/a-Syn
EPR was performed to check the generation ofradical species. It has been reported that DOPA (3,5-dihydroxyphenylalanine) incubated with DNA in thepresence of redox-active transition metal ions, such asFe31, Cu21, or Cr61, induces oxidative damage to targetmolecules via formation of the DNA-DOPA-SQ-�(SQ 5 semiquinone) radical complex, resulting in theproduction of ROS (Pattison et al., 2002). a-Syn can beconsidered a redox-active copper-binding protein(Davies et al., 2011), so a similar free-radical-generating
reaction mechanism may exist (Wang et al., 2010). Fig-ure 5 displays EPR spectra obtained from a-Syn, DA,Cu21, and their mixtures in phosphate buffer. As can beseen in Figure 5, no EPR spectroscopic signals areobserved without Cu21. a-Syn incubated with Cu21
induced a broad and rather weak EPR feature (Fig. 5C)at 3,000–3,400 G, arising from free Cu21; Cu21 isthought to interact with buffer and with a-Syn. The gregion is obscured by a low signal-to-noise ratio.Detailed continuous (CW) and pulsed EPR spectroscopicstudies of Cu21 binding a-Syn have led to proposals ofprotein-based binding modes (Drew et al., 2008; Davieset al., 2011). A comparison with the previous EPR spec-tra of Cu21-bound a-Syn indicates that the EPR spec-troscopic feature, marked in Figure 5C by squares,originates from Cu21 bound to a-synuclein (Drew et al.,2008; Davies et al., 2011). The EPR spectrum (Fig. 5D)of the DA and Cu21 solution gives the sum of twoCu21 signals, one marked In Figure 5 by circles with val-ues for g 5 [2.077 2.077 2.390] 6 0.005 and ACu 5 [2020 390] 6 10 MHz, and the other marked by asterisksdenoting values for g 5 [2.062 2.062 2.308] 6 0.005 andACu 5 [20 20 490] 6 10 MHz.
EPR features marked by circles are assigned toCu21 in buffer (Supp. Info. Fig. 3); features indicated byasterisks are newly detected attributes of Cu21 coordina-tion with DA (for detailed analysis and simulations seeSupp. Info. Figs. 3, 4). a-Syn incubated with DA andCu21 gave the EPR signal shown in Figure 5E, summing
Fig. 4. a: SDS-PAGE result of aggregation of a-Syn (1 equiv., 7 lM)with DA and copper ions under anaerobic conditions. b: Calculatedrelative intensity. Experiments were repeated three times (a shows oneamong three experiments). Error bars indicate standard deviation val-ues. Incubation conditions were 37�C, 16 hr, sodium phosphatebuffer (10 mM, pH 7.4). [Color figure can be viewed in the onlineissue, which is available at wileyonlinelibrary.com.]
Fig. 5. EPR spectra obtained from the frozen solutions of a-syn (30lM; A), DA (150 lM; B), a-Syn (30 lM) 1 Cu21 (150 lM; C),DA (150 lM) 1 Cu21 (150 lM; D), a-Syn (30 lM) 1 DA (150lM) 1 Cu21 (150 lM; E), and a-Syn (30 lM) 1 DA (150lM) 1 Cu21 (150 lM; F) incubated for 11 days. Sodium phosphatebuffer (10 mM, pH 7.4).
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the signals of Cu21 in buffer, Cu21 interacting with DA,and protein-bound Cu21. When the sample was incu-bated for longer times (11 days, room temperature), EPRsignals arose with a diminished Cu21 intensity, and a nar-row radical signal at g 5 2.008 (marked by a triangle; Fig.5F). This observation suggests that Cu21 sites in a-Syn-1 DA 1 Cu21 systems are reduced to Cu1 by a-Synand/or DA to generate an anionic radical, analogous tothe reaction in the tertiary complex of DNA-DOPA-Cu21 (Davies et al., 2011). This suggests that radical-mediated oligomerization may occur slowly, relative tocross-linking, and explains the previous oligomerizationby DA/Cu21 in anaerobic conditions.
Additionally, we performed NBT assays, which stainredox proteins purple (Fig. 6; Paz et al., 1991; Akagawaet al., 2006). Redox reactions were shown in the higholigomer regime, induced from coincubation of a-Syn, DA,and Cu21. This result indicates that radical reactions, induced
by DA/Cu21, may occur in oligomers of a-Syn that thenmake covalent bonds between various types of oligomers.
Cellular Toxicity in SH-SY5Y DopaminergicNeuronal Cell Line in Normal and HypoxicConditions
Finally, we performed a cellular toxicity assay via theWST-1 method to make determinations about the effectsof dopamine and Cu21 (Fig. 7). Comparison experimentsunder aerobic conditions (Supp. Info. Fig. 5; under oxygenconditions) and hypoxic conditions (absence of oxygen)were carried out for comparison. Although the morphol-ogy of some SH-SY5Y cells changed to round underhypoxic conditions, the cells survived for at least 24 hr
Fig. 6. Left: NBT staining of a-Syn (1 equiv., 33 lM) with DA (1.25 equiv.) and Cu21 (62.5equiv.). Right: Intensities of darkness. The double-headed arrow at left indicates purple staining inthe high-oligomer region (tetramer, multimer). Lane a: a-Syn: DA (1:1.25); lane b: a-Syn:-DA:Cu21 (1:1.25:62.5). Incubation conditions were 37�C, 16 hr, sodium phosphate buffer (10mM, pH 7.4). [Color figure can be viewed in the online issue, which is available atwileyonlinelibrary.com.]
Fig. 7. Cellular toxicity in SH-SY5Y dopaminergic neuronal cellsunder normal and hypoxic conditions. Experiments were repeatedthree times. Error bars show standard deviation. P< 0.01, t-test. Neu-romelanin was synthesized from incubation of DA (10 mM) withCu21 (100 mM) ions at room temperature for 6 hr (diluted 1/1,000).
Fig. 8. Transformation of DA into other forms and the neuromelaninform in the presence of oxygen. Adapted from Bisaglia et al. (2007).
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under hypoxic conditions. Under normal conditions, DA/Cu21 showed higher toxicity than DA-only or Cu21-onlycases, which means that the complex form of DA/Cu21
showed more toxicity. Under hypoxic conditions, DA/Cu21 or Cu21 only showed similar toxicity. Almost allcases showed more toxicity under hypoxic conditions thanunder ambient conditions.
When Cu21 was used in cell cultivation media, gen-erally more toxicity was shown for the trials underhypoxic conditions (P< 0.01), whereas, with DA, onlylow toxicity was evident. When DA was coincubatedwith copper ions, cellular toxicity was increased. How-ever, toxicity was similar to cases in which copper wasused. It was discussed above how copper ions are presentin the cytoplasm of the cell; they are able to attach to,
e.g., carboxylates, amino groups, without DA, to mediateredox reaction to produce radical species. Interestingly,when we treated the cells with neuromelanin formed invitro by incubation of DA with Cu21, cellular toxicitywas the highest among these samples. Neuromelanincombined with copper ions may be optimal for directredox reactions, which contributes the highest toxicitylevels. From these results, it was determined that DA/Cu21 ions are important for cellular toxicity in theabsence or presence of oxygen.
DISCUSSION
This study attempts to elucidate the oligomerizationmechanism among DA, Cu1/21, and a-Syn in the
Fig. 9. Illustration of two a-Syn oligomerization mechanisms involving DA and Cu21. a: Cartoonshowing the cross-linking mechanism with reversible bonding. b: Radical-mediated oligomerizationmechanism. Adapted from Linert et al. (2006). The structure of a-Syn was downloaded from theNCBI website (NCBI web site, http://wwwncbinlmnihgov/Structure/mmdb/mmdbsrvcgi?uid531456, MMDB ID: 31456, PDB ID: 1XQ8; Ulmer et al. 2005) and adapted to its presentform. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
366 Ha et al.
Journal of Neuroscience Research
presence or absence of oxygen. In the presence of oxy-gen, DA changed into quinone, chrome, and melaninforms (Fig. 8), which induced the oligomerization of a-Syn in a dose-dependent manner as evidenced by the for-mation of a dark precipitate.
Therefore, DA and DA-neuromelanin attached theoligomer of a-Syn to induce the high position of dimersand trimers. However, when copper was added to thesolution after 6 hr of incubation with DA and a-Syn, DAappeared to detach from the a-Syn oligomers. That meansthat DA attaches noncovalently, or through reversiblecovalent bond formation, to the oligomer of a-Syn. Oli-gomerization induced by DA was reversible in the pres-ence of Cu21.
In the absence of oxygen, a small extent of aggrega-tion was found relative to results for aerobic conditions. Asmall amount of dark precipitate formed even after 72 hr,which means that DA did not change into other forms,and less a-Syn aggregation occurred. However, whenCu1/21 was added, aggregation of a-Syn increased toaerobic levels. Thus, Cu ions are clearly involved in redoxreactions. This is especially evident when copper ions arefirst with other ligands, for example, side groups of a-Synor catechol of DA; it is known that reduction potential isincreased (Wang et al., 2010).
When Cu is attached to catechol groups of DA, itsreduction potential of DA becomes higher, and reductionof DA occurs more easily. In the absence of oxygen, DAattached by its catechol groups may be reduced and maymediate redox reactivity in order to induce radicals detect-able with EPR spectroscopic analysis. Therefore, it is rea-sonable to assume that, according to the data, there are twokinds of oligomerization mechanisms among DA, Cu, a-Syn: 1) a noncovalent (or reversibly covalent) cross-linkingmechanism and 2) a radical-mediated oligomerizationmechanism (Fig. 9). DA alone can induce oligomerizationof a-Syn via noncovalent linking. However, in the pres-ence of DA/Cu21 with the presence or absence of oxygen,radical species can be generated and induce oligomerizationwithout increasing the mass of oligomer, unlike the massincrease of DA moieties alone.
CONCLUSIONS
This study has focused on the interplay among DA, Cuions, and a-Syn in protein aggregation. From all the datafound here, the possibility exists of two different oligome-rization mechanisms being at play that help to explainthese observations. We determined that oligomersinduced by DA can be reversible. We discussed how DAis transformed, most likely into a form resembling DA-neuromelanin, which can induce cross-linking oligomeri-zation with noncovalent bonds. Also, in the presence ofcopper ions, redox reactions by DA/Cu21 can generateradical species, possible in the presence or absence of oxy-gen. DA/Cu21 induces radical-mediated oligomerization.DA and Cu21 are very important components for under-standing PD. Furthermore, when DA and/or Cu21 ionswere used in SH-SY5Y cells, DA/Cu21 under normal
condition showed higher toxicity than Cu21 only,whereas Cu21 and DA/Cu21 showed similar toxicityunder hypoxic condition, which means that redox mech-anism of copper ions may contribute greatly to cellulartoxicity. Therefore, we have an understanding of thesetwo mechanisms: cross-linking with noncovalent bondand radical-mediated oligomerization is a new develop-ment in PD research, and such reactions can help to elu-cidate further certain mechanisms that are biologicallyavailable and possibly connected to the chemical etiologyof PD.
ACKNOWLEDGMENTS
TEM images were obtained through the support of theKorea Basic Science Institute (KBSI). The manuscript waswritten through contributions from all authors. Allauthors have given approval to the final version of themanuscript.
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