Smoking and Parkinson's disease: Does nicotine affect -synuclein fibrillation?

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    Accepted 29 September 2008

    Keywords:-synucleinIntrinsically disordered proteinSmoking

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    neurodegenerative diseases, including Parkinson's disease (PD). Epidemiological studies have shown that

    Biochimica et Biophysica Acta 1794 (2009) 282290

    Contents lists available at ScienceDirect

    Biochimica et Bi

    j ourna l homepage: www.e lsdisorder after Alzheimer's disease. PD is a slowly progressiveneurodegenerative disease caused by the loss of nerve cells in a partof the mid-brain known as the substantia nigra. The cells in the sub-stantia nigra are responsible for dopamine production, a chemicalmessenger involved inmovement coordination. PD is developedwhenthese cells are damaged or destroyed causing different signs of PD:resting tremors, slowness of movement, stiffness of the limbs andbalance problems [1]. The surviving nigral dopaminergic neuronscontain specic proteinaceous inclusions, Lewy bodies (LB) and Lewyneurites (LN), which are also found in several neurodegenerative

    The precise molecular mechanism that leads to the death of thecells in the substantia nigra is still unknown. However, substantialevidence has suggested that the brillation of -synuclein is a criticalstep in the pathogenesis of PD (reviewed in [49]). -synuclein is amajor brillar component of Lewy bodies and Lewy neuritis [10]. It is atypical natively unfolded protein, which possess little orderedstructure at physiological conditions [6,7,9,11,12]. synuclein con-tains 140 amino acid residues and lacks both cysteine and tryptophanresidues [13]. The sequence of -synuclein is divided into threeregions (Scheme 1):diseases [2,3]. It is estimated that 1.5 millio

    Corresponding author. Center for ComputationalDepartment of Biochemistry and Molecular Biology,Medicine, 410 W. 10th Street, HS 5009, Indianapolis, IN6448; fax: +1 317 278 9217.

    E-mail address: vuversky@iupui.edu (V.N. Uversky).1 Prof. Anthony L. Fink has passed away on March 2,

    1570-9639/$ see front matter 2008 Elsevier B.V. Aldoi:10.1016/j.bbapap.2008.09.026common ageing-relatedmon neurodegenerative

    age 50, PD is generally considered as an aging-related disease, andapproximately one of every 100 persons over the age of 65 in the USsuffers from this disorder [1].Parkinson's disease (PD) is the mostmovement disorder and second most com1. IntroductionParkinson's diseaseNicotineHydroquinoneFibrillationMisfoldingare neuro-protective. The brillation of -synuclein was studied in relation to ve different compoundsfound in cigarette smoke: anabasine, cotinine, hydroquinone, nicotine and nornicotine. Thioavin T assays,gel electrophoresis, size exclusion chromatographyhigh performance liquid chromatography (SECHPLC)and atomic force microscopy (AFM) were utilized to monitor the rate of -synuclein brillation and theinhibitory effects of the cigarette smoke components. We show that nicotine and hydroquinone inhibit -synuclein bril formation in a concentration-dependent manner, with nicotine being more effective. TheSECHPLC data show that nicotine and hydroquinone stabilize soluble oligomers. The morphology of theoligomers stabilized by nicotine was evaluated by AFM, which showed the presence of three stable oligomerswith an average height of 16 nm, 10 nm and 4 nm. Comparable results were obtained for the effect of thecigarette smoke components on the A53T mutant brillation. These results show that nicotine andhydroquinone inhibit -synuclein brillation and stabilize soluble oligomeric forms. This information can beused to understand the molecular mechanism of the nicotine and hydroquinone action to developtherapeutic solutions for PD.

    2008 Elsevier B.V. All rights reserved.

    by PD. Since only a small percentage of patients are diagnosed beforeAvailable online 25 October 2008

    smoking can lessen the incidence of Parkinson's disease, indicating that smoke may contain chemicals thatArticle history:Received 24 July 2008

    -synuclein is a small presyfunction is unknown, the aggregated form of -synuclein is a pathological hallmark of severalSmoking and Parkinson's disease: Does n

    Dong-Pyo Hong a, Anthony L. Fink a,1, Vladimir N. Uva Department of Chemistry and Biochemistry, University of California at Santa Cruz, Santab Center for Computational Biology and Bioinformatics, Department of Biochemistry and MIndiana University School of Medicine, Indianapolis, IN 46202, USAc Institute for Biological Instrumentation, Russian Academy of Sciences, 142290 Pushchino,

    a b s t r a c ta r t i c l e i n f o

    Received in revised form 9 September 2008n Americans are affected

    Biology and Bioinformatics,Indiana University School of46202, USA. Tel.: +1 317 278

    2008.

    l rights reserved.otine affect -synuclein brillation?

    ky b,c,z, California 95064, USAcular Biology, Institute for Intrinsically Disordered Protein Research,

    scow Region, Russia

    tic protein (14,460 D) that is abundantly distributed in the brain. Although, its

    ophysica Acta

    evie r.com/ locate /bbapap(1) The N-terminal region, which represents residues 160,contains 11-amino acid imperfect repeats with a consensus motif(KTKEGV).

    (2) The central region, which represents residues 6195, contains ahighly amyloidogenic NAC region and two additional repeats.

    (3) The C-terminal region, which represents residues 96140, isrich in acidic residues and prolines, which are suggested to adopt adisordered conformation [14].

  • smokers which suggests that nicotine exposure prevents againstneuronal insults [23]. Furthermore, nicotine administered orally bygum or transdermally by the patch has been shown to improvesymptoms of Parkinson's disease [2426]. Intriguingly, it has been

    Scheme 1. -synuclein sequence. The three sites of early-onset PD-linked mutations (positions 30, 46 and 53) are highlighted.

    283D.-P. Hong et al. / Biochimica et Biophysica Acta 1794 (2009) 282290The competing kinetic pathways for the aggregation of -synuclein begin with a natively unfolded -synuclein monomer,which partially folds into an aggregation-prone intermediate [12].Depending on the conditions, this intermediate can form threedifferent products: soluble oligomers, insoluble amorphous aggre-gates or insoluble brils [6,7,9] (Scheme 2). The state that -synucleinadopts depends on changes in the environmental conditions such asdecrease in pH, or temperature increase, evolving to the formation ofthe partially folded intermediate [6,7,9,12].

    The involvement of -synuclein in PD was suggested by previousstudies of families with autosomal dominant early-onset of familial PD(FPD) in which three separate mutations in the -synuclein geneA53T, A30P and E46K were linked to the disease [1517]. All three FPDmutations alter the rate of -synuclein aggregation in vitro, but onlythe A53T mutation accelerates bril formation as compared to thewild type proteins, whereas A30P and E46K increase rates ofamorphous aggregation [1820].

    It is believed thatmany environmental and genetic factors affect-synuclein brillation. Factors that accelerate the -synuclein brilla-tion include certain pesticides, metals, lipids, membranes, polycations,glycosaminoglycans (GAGs), and macromolecular crowding [6,7,9].This acceleration is due to the conditions that increase theconcentration of the amyloidogenic intermediate. Oxidative modica-tion was suggested to play an important role in the PD pathogenesisand one of the major factors triggering -synuclein aggregation wasshown to be the formation of free radicals (e.g., see [21]).

    On the contrary, inhibition of brillation occurs when themonomer or non-brillogenic oligomers are stabilized [6,7,9]. Somecompounds in cigarette smoke might cause such inhibition, asepidemiological studies revealed that the smoke compounds sig-nicantly decrease the risk of PD [22,23]. In cigarette smoke, out of themore than 3800 identied compounds, anabasine, cotinine, hydro-quinone, nornicotine, and especially nicotine, potentially representsuch inhibitory compounds. Nicotine is a good candidate forinvestigation because of several reasons. First, nicotine stimulatesstriatal dopamine neurons that are damaged in PD [23]. Second,epidemiological studies have shown that PD is less prevalent inScheme 2. Multiple pathways for -synuclein aggregation. Nu represents the nativelyunfolded -synuclein monomer.reported that nicotine possesses both pro-oxidant and antioxidantproperties [27]. It has been pointed out that because of the nicotine'sbenecial role in the treatment of certain neurodegenerative diseases,its involvement in free radical production or its ability to act as anantioxidant requires careful study to further evaluate its overalltherapeutic usefulness [27]. In addition to nicotine, numerous agentsin tobacco products could modulate biological functions and thedevelopment of PD [23].

    In this study, the effect of nicotine, and three structurally similarcompounds, anabasine, cotinine and nornicotine (Scheme 3), on -synuclein brillation were investigated. The goal was to understandhow these compounds affect the rate of brillation. The results werecompared to those obtained for hydroquinone, a known inhibitor of-synuclein brillation, which is structurally different from the foursmoke compounds (Scheme 3). Thioavin T (ThT) assays, gelelectrophoresis, SECHPLC and atomic force microscope (AFM) wereused to investigate the inhibitory effects. Our results revealed thatnicotine and hydroquinone show strong inhibitory effects on -synuclein brillation by stabilizing oligomers. These ndings mayshed light on novel therapeutic solutions for PD based on the structureof the compounds.

    2. Materials and methods

    2.1. Materials

    The chemical compounds such as nicotine and cotinine wereobtained from Sigma, hydroquinone was purchased from AcrosOrganics, whereas nornicotine and ThT were from Fluka.Scheme 3. Chemical structures of nicotine, cotinine, anabasine, nornicotine, andhydroquinone.

  • 284 D.-P. Hong et al. / Biochimica et Biophysica Acta 1794 (2009) 2822902.2. Expression and purication of human recombinant -synuclein

    Human wild type -synuclein was expressed using E. coli BL21(DE3) cell line transfected with pRK172/-synuclein plasmid (gener-ously donated by M. Goedert, MRC Cambridge). Expression andpurication of human recombinant -synuclein and its mutant fromE. coli were performed as previously described [28]. Culture askscontaining 1 l of LB media were inoculated with 6 ml of a night pre-culture each and subsequently incubated at 37 C at 250 rpm until themedia reached A500=0.91. The culture was then induced withisopropyl-b-D-thiogalactopyranoside (IPTG) and incubated foranother 5 h . The cells were collected by centrifugation at4000 rpm for 15 min at 4 C. The cell pellets were frozen and keptovernight at 20 C. On the next day, pellets were thawed at roomtemperature and redissolved in 40 ml of lysis buffer (50 mM NaCl,20 mM TrisHCl, 0.10% Triton- 100, 0.20 mM phenylmethylsulfonyuoride (PMSF) pH 7.5).

    Sonication at 60% power for 3.5 min total with seven 30 secondsonication bursts and 40 s of rest in between each burst was used tolyse the cells. Subsequently, ammonium sulfate was slowly added toachieve 30% saturation with vigorous stirring on ice. 10 min of gentlestirring completed the precipitation. In order to precipitate the celldebris, the solution was centrifuged at 13,000 rpm for 15 min at 4 C.The supernatant was separated from the pellet and the pellet wasdiscarded. The supernatant was brought to 50% saturation withammonium sulfate. The solution was centrifuged at 13,000 rpm for15 min at 4 C. The supernatant was separated from the pellet andthen discarded. The pellet was saved, dissolved in 50 ml of 10 mMTrisHCl (pH 7.5) and dialyzed against 4 l of 50 mM NaCl, 20 mMTrisHCl (pH 7.5) for 2 h. The dialysis buffer was changed after 2 hand then changed three more times overnight. In order to purify theprotein, the sample from the dialysis was loaded onto a DEAE-Sepharose Fast Flow column previously equilibrated with 5-bedvolume of 50 mM NaCl, 20 mM TrisHCl, and pH 7.2. About 80fractions were collected, and the presence of -synuclein wasveried by using SDS-PAGE. In order to remove the salt from theprotein, the fractions that contained -synuclein were dialyzedagainst deionized water for at least 36 h at 4 C. After the dialysis, theprecipitate was removed by centrifugation. Protein purity evaluatedby mass-spectrometry, SECHPLC and SDS-PAGE was close to 98%.The protein concentration was then determined by A275. The proteinwas frozen in liquid nitrogen and then lyophilized.

    2.3. Fibrillation of WT -synuclein and the ThT assay

    The lyophilized -synuclein was dissolved in 1 ml of 0.02 MNaPO4 (Pi) buffer, 0.1 M NaCl and 0.01% NaN3, pH 7.5. In order toremove any insoluble material, the sample was centrifuged in thecold room for 30 min at 13.2 rpm. The supernatant was thenanalyzed by UV spectrophotometry to estimate protein concentra-tion and by mass-spectrometry, SECHPLC and SDS-PAGE toevaluate the presence of aggregated material. At the beginning ofthe brillation studies, protein samples were predominantly mono-meric and did not contain noticeable amounts of any oligomericforms. Assay solutions contained -synuclein at a concentration of1.0 mg/ml, 20 M ThT with various concentrations of smokecompounds as indicated. A volume of 150 l of the mixture waspipetted into a well of a 96-well plate (white plastic, clear bottom),and a 1/8th in diameter. Teon sphere (McMaster-Carr, Los Angeles)was added. Each sample was run in triplicate or quadruplicate. Theplates were sealed with Mylar plate sealers (Dynex). The plate wasloaded into a uorescence plate reader (Fluoroskan Ascent) andincubated at 37 C with shaking at 600 rpm with a shaking diameterof 2 mm. The uorescence was measured at 30 min intervals withexcitation at 450 nm and emission at 485 nm, with a sampling time

    of 100 ms.The data were t to a sigmoidal curve described by the empiricalequation [29] using SigmaPlot software:

    F = Fi +mit +Ff +mf t 1 + e

    tt50

    1

    where F is the uorescence intensity and t50 is the time to 50% ofmaximal uorescence. The initial baseline during the lag time isdescribed by Fi +mit. The nal baseline after the growth phase hasended is described by Ff +mft. The apparent rate constant, kapp, for thegrowth of brils is given by 1/, the lag time is calculated as t50 2and the amplitude, amp, is given by Ff Fi. Although Eq. 1 gave verygood ts for the ThT kinetic proles, the expression is strictly a simpleempirical means of providing kinetic parameters for comparing ratesof brillation from different samples and does not directly reect theunderlying complex kinetic scheme.

    2.4. SDS-PAGE

    Aliquots of the supernatant were airfuged for 30 min at 20 psi(75,000 rpm) at the end of the brillation. 3 l of the supernatant and1 l of staining gel were added into an Eppendorf tube, boiled for5 min and spun down at 14,000 rpm for 1 min. The amount of solubleprotein present in the supernatant was monitored using theCoomassie-blue staining of the sodium dodecyl sulfate-polyacryla-mide gel electrophoresis (SDS-PAGE) gels.

    2.5. SECHPLC

    Oligomers and monomers were separated using SECHPLC. Afterincubation, an aliquot of the sample was removed. The samples wereloaded onto a BioSep-SEC 2000 column in 50 mM phosphate/100 mMNaCl (pH 7.0) buffer. The samples were eluted at a ow rate of 0.7 ml/min using a Waters 2695 separations module and the presence ofprotein in the sample was evaluated by UV absorption.

    2.6. AFM measurements

    Aliquots of 5 l of the sample and 5 l of 1 M NaCl were placed on amica plate. The mica plate was left to incubate for 24 h and then wasrinsed with water to remove salt and any proteins not bound to themica. The mica plate was then dried with N2. The atomic forcemicroscopic (AFM) images were obtained using a PicoScan Plusmicroscopy, which was equipped with the MAC mode. For the MACmode imaging, probes with a 2.8 N/m spring constant and a 75-kHzresonance frequency were used. A scan rate of 0.51 line/s with 512data points per line, at a driver current of 105 were used. Theheight range of 1.0 to 100 was estimated by section analysis. At leastfour regions of the mica surface were examined to verify that similarstructures existed throughout the sample.

    3. Results

    3.1. Nicotine and hydroquinone inhibit the brillation of -synuclein

    The kinetics of -synuclein bril formation was monitored by thecharacteristic increase in the ThT uorescence intensity. ThT is auorescent dye that interacts preferentially with the cross--enriched amyloid-like brils and is commonly used to detect theamyloid brils and to follow the brillation process. ThT binds to -sheet and non--sheet cavities with the diameters of 89 [30]. ThTuorescence is an efcient method to monitor brillation kineticsbecause the brils of -synuclein contain cross- laments thatspecically interact with ThT. As more brils are formed, there is anincrease in ThT uorescence intensity. The kinetics of -synuclein

    brillation is represented by a sigmoidal curve, which begins with a

  • lag phase, followed by a growth phase, and ends with an equilibriumphase.

    Fig. 1 demonstrates the time-dependent changes in the ThTuorescence intensity during the -synuclein brillation in theabsence or presence of ve smoke compounds at the same

    concentration of 200 M. This graph shows that anabasine, nornico-tine and cotinine do not affect the kinetics of bril formation ascompared to the control. It also illustrates hydroquinone and nicotinehas a noticeable inhibitory effect on the brillation. Nicotine shows amuch stronger inhibition, which is reected in longer lag-time and inthe smaller ThT signal.

    Fig. 2 shows the time-dependent changes in the ThT uores-cence intensity during the -synuclein bril formation as afunction of different concentrations (100 M, 200 M and400 M) of cotinine (Fig. 2A), anabasine (Fig. 2B), nornicotine(Fig. 2C), hydroquinone (Fig. 2D) and nicotine (Fig. 2E) as comparedto the control. Fig. 2AC show that even at a concentration of400 M cotinine, anabasine and nornicotine did not affect the -synuclein brillation. On the other hand, Fig. 2D,E demonstrate thatas the hydroquinone and nicotine concentration increases, the ThTuorescence signal decreases, suggesting the effective inhibition ofthe -synuclein brillation. Compared to hydroquinone, nicotine isa slightly better inhibitor as reected in lower ThT signal andslower brillation kinetics.

    3.2. Hydroquinone- and nicotine-induced stabilization of the oligomericform of -synuclein: SDS-PAGE analysis

    To conrm the inhibitory effect of hydroquinone and nicotine onthe -synuclein brillation, the samples at the end of the incubationperiod were checked by SDS-PAGE. Incubated samples were

    Fig. 1. Effect of different cigarette smoke compounds on the brillation kinetics of -synuclein: -synuclein (1 mg/ml) alone (black circles); -synuclein in the presence of200 M cotinine (red triangles); 200 M anabasine (green squares); 200 Mhydroquinone (yellow diamonds); 200 M nicotine (blue triangles); and 200 Mnornicotine (pink pentagons).

    285D.-P. Hong et al. / Biochimica et Biophysica Acta 1794 (2009) 282290Fig. 2. Effect of different concentrations of various smoke compounds on the brillatiohydroquinone, (E) nicotine. Black circles, 0 mM; red triangles, 100 mM; green squares, 200n kinetics of 1 mg/ml -synuclein. (A) cotinine, (B) anabasine, (C) nornicotine, (D)

    mM; yellow diamonds, 400 mM.

  • the second peak (10 nm), and higher than the height of the oligomersin the third peak (4 nm).

    3.5. Nicotine and hydroquinone inhibit the -synuclein A53Tmutant brillation

    The early onset of familial PD is associated with the differentmissense mutations in the -synuclein gene, corresponding to A53T,A30P and E46Kmutations in the-synuclein protein. Therefore, at thenext stage, we evaluated the effect of hydroquinone, nicotine andnornicotine on brillation of the A53T mutant. Fig. 6 represents thetime-dependent changes in the ThT uorescence intensity during theA53T bril formation in the presence of different compounds at aconcentration of 100 M. In agreement with earlier data, Fig. 6 showsthat the A53T mutant brillates faster than the wild type protein.A53T brillation was not affected by nornicotine, whereas hydro-quinone and nicotine possessed an inhibitory effect. Similar to thewild type protein, nicotine was a better inhibitor of the A53Tbrillation.

    Fig. 7A compares the effects of various nornicotine concentrationson the A53T brillation and shows that this compound did not effectbrillation even at 400 M concentration. On the other hand, Fig. 7B,Cshow that hydroquinone inhibited the A53T brillation on a

    286 D.-P. Hong et al. / Biochimica et Biophysica Acta 1794 (2009) 282290subjected to the centrifugation to pellet the mature brils and theresulting supernatants were used for the SDS-PAGE analysis (Fig. 3).This analysis was carried out for all the hydroquinone concentra-tions. Fig. 3 clearly shows that soluble protein is present in thesupernatant of -synuclein incubated in the presence of all thehydroquinone concentrations. In these cases, in addition to the bandcorresponding to the monomeric -synuclein, an oligomeric form ofthe protein was also detected (see lines 35 in Fig. 3). This indicatesthat non-brillar -synuclein is present in the supernatant,suggesting that the brillation of -synuclein is effectively inhibitedby hydroquinone. Similar results were also obtained for nicotine(data not shown).

    3.3. Hydroquinone- and nicotine-induced stabilization of theoligomeric form of -synuclein: SECHPLC analysis

    In order to gain further information on the oligomeric state of -synuclein incubated in the presence of hydroquinone and nicotine,SECHPLC analysis was performed for samples incubated withdifferent concentrations of these compounds. Fig. 4A shows thatcompared to the control, most of the -synuclein in the presence ofnicotine or hydroquinone is in a form of higher molecular weightoligomers. Fig. 4B compares the elution proles of the monomeric -synuclein with the protein samples incubated in the presence of400 M nicotine or hydroquinone. Fig. 4B clearly shows that severaldifferent oligomer types were formed in the presence of nicotine andhydroquinone. Interestingly, although these smoke compounds seemto stabilize similar oligomers, the relative populations of theseoligomers were different for samples containing nicotine andhydroquinone. In addition to oligomers, hydroquinone samplescontained noticeable amount of the monomeric -synuclein asevidenced by the presence of the measurable shoulder in the

    Fig. 3. Determination of the concentration of -synuclein in the supernatant using SDS-PAGE. (1) -synuclein, 1 mg/ml; (2) control experiments where -synuclein wasbrillated alone, (3) -synuclein after brillation in the presence of 100 Mhydroquinone; (4) -synuclein after brillation in the presence of 200 M hydro-quinone; and (5) -synuclein after brillation in the presence of 400 M hydroquinone.corresponding elution prole.

    3.4. Nicotine-induced stabilization of the oligomeric form of -synuclein:AFM analysis

    Since the nicotine sample contained a higher amount of oligomersthan the hydroquinone sample (this conclusion follows from the factthat SECHPLC prole for 400 M hydroquinone preserves a smallshoulder at the monomer peak position (see Fig. 4B), indicating thatsome monomers are still present), each of the three peaks of the -synuclein sample containing the 400 M nicotine (Fig. 4B) weresubjected to the AFM imaging in order to evaluate the size and theshape of these oligomers. Fig. 5 represents the results of this analysisboth as AFM images and the correspondingheight histogram. Fig. 5ACcorrespond to the samples from the rst, second and third SECHPLCpeaks respectively. As expected, the average height of the oligomers inthe rst peak (16 nm)was greater than the height of the oligomers inFig. 4. SECHPLC demonstration that nicotine and hydroquinone stabilize -synucleinoligomers. (A) -synuclein (1 mg/ml) alone or incubated in the presence of 100 M,200 M, 400 M nicotine, and 100 M, 200 M, and 400 M hydroquinone. (B) -synuclein (1 mg/ml, 1) alone or incubated in the presence of 400 M nicotine (2) or

    400 M hydroquinone (3).

  • Fig. 5. AFM images and height distributions of an -synuclein sample containing 400 M nicothe SECHPLC column (Fig. 4B).

    Fig. 6. Effect of different smoke compounds on the brillation kinetics of the A53Tmutant. Mutant -synuclein incubated at 1 mg/ml alone (black circles) or in thepresence of 100 M nicotine (red triangles); 100 M hydroquinone (green squares); or100 M nornicotine (yellow diamonds).

    287D.-P. Hong et al. / Biochimica et Biophysica Acta 1794 (2009) 282290concentration-dependent manner, whereas the brillation processwas completely inhibited in the presence of the smallest nicotineconcentration (100 M).

    3.6. Stabilization of the oligomeric form of the A53T -synuclein withhydroquinone and nicotine

    SDS-PAGE carried out for A53T samples incubated in the presenceof the different concentrations of hydroquinone revealed that theA53T mutant protein was in the supernatant (Fig. 8). Addition ofnicotine produced similar inhibitory effect (data not shown). Thedistribution of the soluble oligomers in A53T samples incubated in thepresence of 400 M of hydroquinone or nicotine was analyzed by theSECHPLC (Fig. 9). As with the wild type proteins, in addition to themonomer, different sized oligomers were detected. Taken togetherthese data suggest that similar to thewild type protein, the brillationof the A53T -synuclein is effectively inhibited by the hydroquinoneand nicotine in a concentration dependent manner. In all cases, thisinhibition occurs due to the preferential stabilization of the solubleoligomers.

    tine. Samples were collected from the rst (A), second (B) or third peak (C) eluting from

  • 4. Discussion

    PD is a neurodegenerative disease occurring in about 1% of thepopulation over the age of 65. It is characterized by the damage of thedopaminergic nigrostriatal neurons that leads to motor impairment.The most commonly used treatment for PD is dopamine replacementtherapy. Although, somewhat effective, the L-dopa only providessymptomatic relief with an unavoidable disease progression. It alsoloses efcacy with time and can cause drug-induced side effects [31].The currently available therapies aim at the improvement of thefunctional capacity of the patient as long as possible but do not affectthe progression of the neurodegenerative processes [32]. All thisclearly emphasizes the need for newer and more effective therapeuticsolutions. This problem is receiving more attention and is beingsubject to extensive research.

    Many epidemiological studies have found that smoking isassociated with a lower incidence of PD [22,23]. The risk of PD innonsmokers is about twice that of smokers. This means that cigarettesmokers are about 50% less likely to have PD and that the patientswithPD are about 50% less likely to have smoked cigarettes during theirlifetime. These ndings are important because, being understood atthe molecular level they may provide some clues about noveltherapeutic strategies for protection against PD.

    cigarette smoke compounds such as nicotine and hydroquinone in aconcentration-dependent manner. This inhibition involves the

    Fig. 8. Determination of the concentration of the soluble A53T mutant in thesupernatant after brillation using SDS-PAGE. (1) mutant, 1 mg/ml, (2) mutant afterincubation alone, (3) A53T after incubation in the presence of 100 M hydroquinone, (4)A53T after incubation in the presence of 200 M hydroquinone, and (5) A53T afterincubation in the presence of 400 M hydroquinone.

    Fig. 9. SECHPLC demonstration that nicotine and hydroquinone, being coincubatedwith the A53T mutant, stabilize oligomers. Mutant A53T; A53T incubated with 400 M

    288 D.-P. Hong et al. / Biochimica et Biophysica Acta 1794 (2009) 282290Fig. 7. Effect of different concentrations of various cigarette smoke compounds on thebrillation kinetics of the 1 mg/ml A53T mutant. (A) nornicotine, (B) hydroquinone, (C)nicotine. Black circles, 0 mM; red triangles, 100 mM; green squares, 200 mM; and

    yellow diamonds, 400 mM.The successful inhibition of -synuclein brillation is believed tobe important for the prevention or control of PD. In this study, weshowed that two compounds of cigarette smoke, nicotine andhydroquinone, led to the efcient inhibition of the -synuclein brilformation in a concentration-dependent manner (Fig. 2). The SECHPLC analysis revealed that instead of the insoluble amyloid-likebrils, three stable oligomeric forms were formed (Fig. 4). The AFMimaging of these oligomers showed that they are mostly sphericalspecies with heights of 16 nm, 10 nm, and 4 nm (Fig. 5). We alsoestablished that nicotine and hydroquinonewere successful inhibitorsof the A53T brillation (Figs. 69). Finally, our data showed thatnicotine was a more effective inhibitor than hydroquinone.

    Our ndings are in a perfect agreement with the results of earlierstudies on the nicotine effect on the -synuclein brillation. In fact,using thioavin S (ThS) it has been shown that the brillation of bothwild type and A53T -synucleins (both at the concentration of140 M) was effectively inhibited by nicotine in a concentrationdependent manner.16 AFM and EM analyses conrmed the ThS dataand clearly showed that nicotine indeed inhibited -synuclein brilformation, as samples of-synuclein (140 M) incubatedwith 100 Mnicotine contained smaller amount of brils and these brils wereshorter and thinner in comparison with those formed in the absenceof nicotine [33]. We have established that nicotine is able to inhibit -synuclein brillation almost completely. Our experimental settingswere quite different from those in the work of Ono et al. [33], as weused smaller -synuclein concentrations (70 M) and higher nicotineconcentrations (up to 400 M).

    Thus, -synuclein brillation can be effectively inhibited bynicotine, or with 400 M hydroquinone.

  • 289D.-P. Hong et al. / Biochimica et Biophysica Acta 1794 (2009) 282290stabilization of soluble oligomeric forms. How can the stabilization ofsuch soluble oligomers be related to the pathology of neurodegenerativediseases? In this respect it is important to remember that for a long time itwas believed that the amyloid brils were harmful. However, a novelemerging paradigm favors the idea that the deposited proteinaceousinclusions (such as senile plaques inAlzheimer's disease or Lewybodies orLewy neurites in Parkinson's disease, etc.) are not cytotoxic. Instead, theformation of some small oligomers, known as various protobrils, isresponsible for the neurotoxicity [3437]. This hypothesis is supported bythe fact that the amount of brillar deposits found at autopsy does nottypically correlate with the clinical severity of Alzheimer's or Parkinson'sdisease [37]. Furthermore, the dopamine-dependent neurotoxicity of -synuclein in PD was shown to be mediated by 5483-kD soluble proteincomplexes that contain -synuclein and 14-3-3 protein, which areelevated selectively in the substantia nigra in PD [21]. Furthermore, inanimal models of PD and AD, the disease-like phenotypes were shown todevelop before the appearance of the brillar deposits [38,39]. Non-brillar oligomers, known as protobrils, of various amyloidogenicproteins (both disease-related and non-disease-associated) are toxic incell cultures and are able to disruptmembrane integrity in vitro [36,37,4043]. Special attention has been paid to the so-called amyloid pores; i.e.,morphologically similar annular protobrils, that resemble a class of pore-forming bacterial toxin, as these oligomers might cause the inappropriatemembrane permealization leading to cell dysfunction and cell death[36,37,4345].

    Although the hypothesis that the small oligomers are cytotoxicand that the larger insoluble aggregates found in Lewy bodies andother proteinaceous inclusions may be protective is very important, itis necessary to remember that protein aggregates (includingoligomers) are highly heterogeneous. This heterogeneity might becaused either by the heterogeneous starting materials or by multiplepathways of assembly, or by both these factors. Therefore, it isdifcult to believe that all the soluble oligomers, with theirastonishing morphological variability, will be cytotoxic in the samedegree. In fact, we have recently shown that the avonoid baicaleinwas able to inhibit the -synuclein brillation via the stabilization ofsoluble oligomers that possessed very specic structural features[46], being spherical in shape (according to the AFM and EM data),having relatively globular structure with packing density intermedi-ate between that of pre-molten globules and typical globular proteins(according to the Kratky plot analysis of the SAXS data), and havingrelatively well-developed secondary structure (according to the FTIRand far-UV CD analysis). Furthermore, these oligomers werecharacterized by high thermodynamic stability and were able toinhibit brillation of baicalein-untreated -synuclein. The mostimportant nding was the fact that these highly stable andbrillation-inhibiting oligomers did not disrupt the integrity of thebiological membrane. Based on these observations it has beenconcluded that the soluble oligomer formation does not alwayscreate harm and can be benecial [46]. We believe that similaroligomers might be stabilized by other chemical compounds (e.g., bythe compounds of the cigarette smoke analyzed on this study), whichmight act similarly to baicalein; i.e., via the specic stabilization ofthe thermodynamically stable, non-brillating soluble oligomers thatcan inhibit -synuclein brillation and do not cause the inappropri-ate membrane permealization.

    Obviously, further studies are needed for the deeper under-standing of the molecular mechanisms of the inhibition of -synuclein brillation by nicotine and other cigarette smoke com-pounds. These studies might also be important for the development ofnovel anti-PD therapeutic strategies. An important question iswhether the inhibition observed by us in vitro also takes place insidethe living cells. If this inhibition does occur in the cell, then does itfollow a receptor-related mechanism or a non-receptor-mediatedmechanism? Another question is whether other chemicals in smoke

    can possess the neuro-protective effect. These and other issues need tobe resolved in order to understand the molecular basis for theneuroprotection caused by smoking.

    5. Conclusions

    We show that compounds of cigarette smoke such as nicotine andhydroquinone are able to inhibit the formation of -synuclein brilsin a concentration-dependent manner, with nicotine being the moreeffective inhibitor. SECHPLC, SDS-PAGE, and AFM analyses revealedthat nicotine and hydroquinone stabilized soluble oligomers. Similardata were obtained for the A53T mutant. These ndings are rst stepsin better understanding the molecular mechanism of the nicotine-and hydroquinone-mediated inhibition of -synuclein brillation,which potentially might help in developing novel therapeuticsolutions for PD.

    Acknowledgements

    This researchwas supported inpart bygrantsR01NS39985 (toD.-P.Hand A.L.F.), R01 LM007688-01A1 (V.N.U.) and GM071714-01A2 (V.N.U.)from the National Institutes of Health and by a grant from the ProgramMolecular and Cellular Biology of the Russian Academy of Sciences (toV.N.U.). We gratefully acknowledge the support of the IUPUI SignatureCenters Initiative.

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    290 D.-P. Hong et al. / Biochimica et Biophysica Acta 1794 (2009) 282290

    Smoking and Parkinson's disease: Does nicotine affect -synuclein fibrillation?IntroductionMaterials and methodsMaterialsExpression and purification of human recombinant -synucleinFibrillation of WT -synuclein and the ThT assaySDS-PAGESECHPLCAFM measurements

    ResultsNicotine and hydroquinone inhibit the fibrillation of -synucleinHydroquinone- and nicotine-induced stabilization of the oligomeric form of -synuclein: SDS-PA.....Hydroquinone- and nicotine-induced stabilization of the oligomeric form of -synuclein: SECHP.....Nicotine-induced stabilization of the oligomeric form of -synuclein: AFM analysisNicotine and hydroquinone inhibit the -synuclein A53T mutant fibrillationStabilization of the oligomeric form of the A53T -synuclein with hydroquinone and nicotine

    DiscussionConclusionsAcknowledgementsReferences

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