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Brain Research Bulletin 75 (2008) 648–654

Research report

Differences between normal and alpha-synuclein overexpressingSH-SY5Y neuroblastoma cells after A�(1-42) and NAC treatment

Akos Hunya a,∗, Istvan Foldi a,1, Viktor Szegedi a,2, Katalin Soos a,3, Marta Zarandi a,3,Antal Szabo a,1, Denes Zadori a,1, Botond Penke a,b,4, Zsolt L. Datki b,1

a Department of Medical Chemistry, University of Szeged, Szeged, Hungaryb Supramolecular Research Group, Hungarian Academy of Sciences, Szeged, Hungary

Received 4 June 2007; received in revised form 27 September 2007; accepted 22 October 2007Available online 20 November 2007

bstract

Alpha-synuclein (�SN) plays a major role in numerous neurodegenerative disorders, such as Alzheimer’s disease and Parkinson’s disease.ntracellular inclusions containing aggregated �SN have been reported in Alzheimer’s and Parkinson’s affected brains. Moreover, a proteolyticragment of �SN, the so-called non-amyloid component of Alzheimer’s disease amyloid (NAC) was found to be an integral part of Alzheimer’sementia related plaques. Despite the extensive research on this topic, the exact toxic mechanism of �SN remains elusive. We have taken thedvantage of an �SN overexpressing SH-SY5Y cell line and investigated the effects of classical apoptotic factors (e.g. H2O2, amphotericin Bnd ruthenium red) and aggregated disease-related peptides on cell viability compared to wild type neuroblastoma cells. It was found that �SNverexpressing cells are more sensitive to aggregated peptides treatment than normal expressing counterparts. In contrast, cells containing elevated

mount of �SN were less vulnerable to classical apoptotic stressors than wild type cells. In addition, �SN overexpression is accompanied byltered phenotype, attenuated proliferation kinetics, increased neurite arborisation and decreased cell motility. Based on these results, the �SNverexpressing cell lines may represent a good and effective in vitro model for Alzheimer’s and Parkinson’s disease.

2007 Elsevier Inc. All rights reserved.

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eywords: Alpha-synuclein; Beta-amyloid; NAC; SH-SY5Y; Alzheimer’s dise

. Introduction

The central pathological feature of Parkinson’s disease (PD)

s the accumulation and aggregation of a synaptic protein, alpha-ynclein (�SN). Several findings underlie its main role, suchs mutations in �SN are associated with dominantly inher-

∗ Corresponding author at: H-6720 Szeged, Szikra utca 2, Hungary.el.: +36 62 546853; fax.: +36 62 546 826.

E-mail addresses: akos.hunya@gmail.com (A. Hunya),stvanfoldi82@yahoo.com (I. Foldi), szegv@yahoo.com (V. Szegedi),oska@mdche.szote.u-szeged.hu (K. Soos),arandimarta@yahoo.com (M. Zarandi), ertpar@freemail.hu (A. Szabo),adorid@freemail.hu (D. Zadori),botond@mdche.szote.u-szeged.hu (B. Penke),atkiz@mdche.szote.u-szeged.hu (Z.L. Datki).1 Tel.: +36 62 546853.2 Tel.: +36 62 546854.3 Tel.: +36 62 545140.4 Tel.: +36 62 545 135.

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361-9230/$ – see front matter © 2007 Elsevier Inc. All rights reserved.oi:10.1016/j.brainresbull.2007.10.035

arkinson’s disease

ted PD and �SN was found to be the predominant protein inewy bodies [20,32,36]. Moreover, �SN fibrillization appears

o be a major event in other neurodegenerative diseases, suchs multiple system atrophy, where �SN is part of glial cyto-lasmic inclusions [37], and it is associated with the neuronalntranuclear inclusions of Huntington’s disease [5,15]. �SN mayven participate in the pathogenesis of Alzheimer’s diseaseAD), because intracellular inclusions containing aggregatedSN have been reported in Alzheimer’s affected brains [19,28].

n addition, a proteolytic fragment of �SN, the so-called non-myloid component of Alzheimer’s disease amyloid (NAC) wasescribed as an integral part of plaques in demented patients [29].

The normal physiologic role of �SN is under extensiveesearch. It was cloned [38] and found to be a human homologf the Torpedo ray synuclein, which had been previously iden-

ified in synaptic vesicle preparations [27]. Taken its abundantxpression in the nervous system and its close association withresynaptic vesicles [7,17], a role in vesicle refilling was sug-ested [1]. Moreover, �SN undergoes a marked conformational

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A. Hunya et al. / Brain Rese

hange upon binding to membranes and interacts with a num-er of microtubule-associated proteins [17], hence, a chaperoneunction in the SNARE complex was put forward [4].

By taking the advantage of an �SN overexpressing cell line,e have investigated the effects of classical apoptotic stressors

nd aggregated neurodegenerative disease-related peptides onSN overexpressing and normal cell lines. It is reported here

hat the �SN overexpressing cell line displayed a higher degreef cell death after application of aggregated beta-amyloid (A�-42) or NAC. In contrast, �SN overexpressing cells were lessensitive to apoptotic agents, compared to its wild-type counter-arts.

. Materials and methods

.1. Materials

Amyloid beta 1-42 (A� 1-42) and non-amyloid component of Alzheimer’sisease amyloid (NAC) were synthesized using solid phase methodologyn our laboratory at the Department of Medical Chemistry, University ofzeged, Hungary. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-ide (MTT), dimethyl-sulfoxide (DMSO), bovine serum albumin (BSA),

ll-trans retinoic acid (RA), 12-O-tetradecanoyl-phorbol-13-acetate (TPA),ydrogen peroxide (H2O2), amphotericin B, ruthenium red, Triton X-100,ween 20, protease inhibitor cocktail, polyclonal anti-�SN antibody pro-uced in rabbit, and mouse anti-�-tubulin monoclonal antibody were obtainedrom Sigma–Aldrich (Budapest, Hungary). Fetal bovine serum (FBS), phos-hate buffered saline (PBS), l-glutamine, penicillin–streptomycin, and MEMon-essential amino acids were purchased from Gibco (Budapest, Hun-ary), 96-well plates from Nunc (Roskilde, Denmark), peroxidase-conjugatedoat anti-rabbit IgG antibody from Dako (Glostrup, Denmark), chemo-uminescent substrate of peroxidase from Pierce (Rockford, IL, USA),nd bicinchoninic acid (BCA) assay kit from Novagen (Madison, WI,SA).

.2. Photomicrographs

Images were taken by using an inverted research microscope, type IX71 ofLYMPUS (OLYMPUS, Budapest, Hungary).

.2.1. Experimental treatments of differentiated cellsThe �SN “normal expressing” (NE) and the �SN “wild type overexpress-

ng” (OE) SH-SY5Y cell lines were obtained from Alex Liu (Department ofeurology, University of Saarland, Homburg, Germany). The cells were grown

o confluency at 37 ◦C in Dulbecco‘s modified Eagle’s medium (MEM):F-121:1) containing phenol red on 96-well plates in a humidified atmosphere of% CO2 for 8 days. l-Glutamine (4 mM), penicillin (200 units/ml), strep-omycin (200 �g/ml), MEM non-essential amino acids and all-trans retinoiccid (RA) with phorbol ester (TPA) [12,23,33] were dissolved in dimethyl-ulfoxide (DMSO). The final concentrations of RA, TPA and DMSO were0 �M, 16 nM and 0.5%, respectively. Fetal bovine serum (10%) was alsodded to the medium. The number of passage in case of both cell lines was. On the first day, the number of non-differentiated cells in the wells was.5 × 105 cells/ml. After 8–10 days of differentiation, the cells were attachedo the plate as a monolayer, and cell counting indicated 3 × 105 cells/cm2

equals to 6.5 × 105 cells/ml in suspension). We have previously done the3H]-thymidine incorporation test using SH-SY5Y NE cells and the resultsere published in Ref. [9]. According to the results of the test, the differ-

ntiation of the SH-SY5Y cell line (treated with RA) was effective, since itisplayed minimal [3H]-thymidine incorporation increase. During the 8-day

ifferentiation, none of the cell types displayed significant cell death (data nothown). Aggregation of A�(1-42) and NAC were performed in aqueous solu-ion (100 �M) by gentle shaking at room temperature for 1 h or 8 days. Inrder to prevent infection, the solutions were ultrasonicated daily. The aggrega-ion states of the peptides are: 1 h pre-aggregated A�(1-42) (protofibrils), 8-day

cceia

ulletin 75 (2008) 648–654 649

re-aggregated A�(1-42) (fibrils) [14], 1 h pre-aggregated NAC (protofibrils)nd 8-day pre-aggregated NAC (fibrils; TEM pictures not shown). The origi-al supernatant solution was removed from the cells with a pipette, and a newedium (100 �l/well, containing 2% FBS and aggregated A�(1-42) or NAC in

0 �M concentration) was quickly added to the wells and was kept at 37 ◦C for4 h.

.3. MTT bioassay

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), aidely used assay was performed to compare the viability of cells [25]. Dif-

erentiated neuroblastoma cells [9] were incubated in a 96-well plate for 24 hith the following peptides: (a) 10 �M of A�(1-42) pre-aggregated for 1 h ordays; (b) 10 �M NAC pre-aggregated for 1 h or 8 days. In a second set of

xperiment, cells were treated with the following classical apoptotic factors:

2O2 (40 �M), amphotericin B (3 �M) and ruthenium red (200 �M). MTTolution was added to each well (0.4 mg/ml), containing 100 �l cell cultureedium, and then the mixture was incubated for 3 h. The MTT solution was

arefully decanted and formazan was extracted from the cells using 100 �l/wellf a DMSO/EtOH (4:1, v/v). The colour intensity of formazan was measuredy a 96-well plate reader (BMG Labtech, Budapest, Hungary) at 550 nm. AllTT assays were triplicated, hence, one measurement contained seven parallels

n = 21).

.4. Western blot analysis

Normal and �SN overexpressing differentiated neuroblastoma cells werextracted in phosphate buffered saline (PBS) containing 1% Triton X-100nd 1% protease inhibitor cocktail. The cell extracts were centrifuged at6000 × g for 10 min and supernatants were saved. 15 �g of total proteinas separated by sodium dodecyl sulfate-polyacrylamide gel electrophore-

is (SDS-PAGE) under reducing conditions according to the method ofaemmli [22]. Proteins were blotted onto nitrocellulose membrane. The blotas incubated overnight in the presence of rabbit anti-alpha-synuclein poly-

lonal antibody diluted 1:500 in PBS. Then, the membrane was washed threeimes in PBS–0.05% Tween and incubated for 2 h peroxidase-conjugatedoat anti-rabbit IgG antibody diluted 1:10000 in PBS. Then the membraneas washed three times in PBS–0.05% Tween. The immune complexesere detected with chemo-luminescent substrate of peroxidase according

o the supplier’s instructions. Protein concentration was determined usingbicinchoninic acid (BCA) assay kit. Images were densitometry analyzed

sing Scion Image software. Densitometric studies were normalized for �-ubulin.

.5. Digital cell motility assay

We used the modified method developed by Ariano et al. [2]. Measure-ents were performed with a light-fluorescence inverted research microscope

t 37 ◦C (using a heated object table). Images were taken by a 24-bit digitalolour-View II FW camera (with CCD arrays of 2080 × 1544 pixels) inter-

ocked with the imaging system of the microscope at 0 (starting point) and0 min after treatment with 10 �M A�(1-42) or 10 �M NAC. A magnificationf 200× was used. Neurites always occupied 50–60% of the area under investiga-ion. Image acquisition and analysis were performed by analySIS 3.2 softwareOLYMPUS, Budapest, Hungary). The images were converted with Reimerigital filter and then were separated by red/green/blue (RGB)-studio func-ion into three different 8-bit monochromatic images. The red or green colourhannel was used as respective monochromatic picture in measure/intensityrofile/horizontal mean function. This function of the analySIS program gives aistogram (data array) of the white colour intensity of the respective monochro-atic images. The histograms of 0 and 30 min images were compared, and the

orrelation coefficient (r) was calculated. The data obtained from the untreatedells (control) were taken as 100%. The following formula was used forxpressing cell motility: CMB(%) = (1 − rB)/(1 − rA) × 100; CM = cell motil-ty, A = untreated cells, B = treated cells, r = correlation coefficient (between 0nd 30 min).

6 arch Bulletin 75 (2008) 648–654

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Fig. 2. Proliferation kinetics of non-differentiated SH-SY5Y neuroblastomacell lines (NE and OE). The proliferation of OE cells was much slower,than that of NE cells. The initial cell number was 2 × 105/ml in both celllines. �SN = alpha-synuclein protein, NE = alpha-synuclein normal express-

50 A. Hunya et al. / Brain Rese

.6. Statistical analysis

Data are presented as mean ± S.E.M. Unpaired Student’s t-test (for Westernlot analysis) and ANOVA with post hoc Bonferroni was used for statisti-al evaluation, both performed by SPSS for Windows 9.0. A p ≤ 0.05 valueas considered statistically significant, unless otherwise stated (e.g. p ≤ 0.01;≤ 0.001).

. Results

.1. Phenotypic characterization of cell lines

Eight days after plating, the two types of SH-SY5Y cellsdifferentiated NE and differentiated OE) showed differentistribution and shape as investigated by transmission lighticroscopy at 200× magnification (Fig. 1). In the case of OE

ells, the cell density was lower, while synaptogenesis and neu-

ite growth was more pronounced than that of the NE cells.eurite lengths of NE cells were increased after differentia-

ion. However, �SN OE cells displayed a marked increase inhe length of cell processes (data not shown).

ig. 1. Snapshots of alpha-synuclein expressing SH-SY5Y cell lines in trans-ission light. (A) Differentiated alpha-synuclein normal expressing (NE) cells;

B) differentiated alpha-synuclein overexpressing (OE) cells. In case of OE cells,he cell density was lower, while synaptogenesis and neurite growth was moreronounced than in the NE cells. Scale bar 30 �m.

ing; OE = alpha-synuclein overexpressing. Differences (*) compared with theut

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ntreated control values (0 min) are significant at a level p ≤ 0.01, n = 21. Sta-istical analysis by ANOVA post hoc test, Bonferroni.

.2. Measurement of proliferation kinetics with MTT assay

In order to follow the kinetics of cell proliferation, we havetilized MTT assay with an initial cell number of 2 × 105/mlrom non-differentiated cells. The kinetics were traced up to 4ays after culturing. Normal expressing cells exhibited a rapidncrease in MTT reduction capacity, which became significantlready on day 2 (n = 21). In contrast �SN OE cells exhibitedslower increase in MTT reduction, and the asymptote of the

urve was 0.6 (compared to 1.2 for the �SN NE cells) (Fig. 2).

.3. Western blot analysis of αSN

The expression level of �SN in differentiated NE and OEells was analysed by Western blot (Fig. 3). In both cell linesmmunoreactive bands were detected at 19 kDa, which indicateshe presence of the wild type form of monomeric �SN proteinFig. 3A). The sample divided from OE cell line contains also a24–26 kDa form of �SN, which refers a covalently modified

soform (Fig. 3A). Two types of covalently modified �SN iso-orm have been reported: one is the O-glycosylated [35] and thether is the monoubiquitinated [18]. After comparison, the �SNrotein content of NE and OE cells we detected a significantlyigher �SN level in OE cells (Fig. 3B and C).

.4. The effects of Aβ(1-42) and NAC on cell viability

Differentiated cells were treated with protofibrillar and fibril-ar A�(1-42) or NAC, and cell viability was measured by MTTssay. Values were expressed as %, where 100% was the non-reated control value (not shown). A�(1-42) in both aggregationtates was toxic to NE cells, i.e., NE cells exhibited 57 ± 5.6%

nd 49 ± 8.3% viability after 1 and 8 days, respectively (n = 21).n contrast, A�(1-42) was even more toxic to OE cells, i.e., OEells exhibited 31 ± 4.9% and 25 ± 7.3% viability after 1 anddays, respectively (n = 21). Regarding the NAC peptide, NAC

A. Hunya et al. / Brain Research Bulletin 75 (2008) 648–654 651

Fig. 3. Comparison of alpha-synuclein levels of normal and alpha-synuclein overexpressing neuroblastoma cell lines. (A) Western blots show representative alpha-s ) ands otein;O

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Bduwthan the NE counterparts (75 ± 5.9% for H2O2, 62 ± 7.9%for amphotericin B and 87 ± 7.1% for ruthenium red, n = 21)

ynuclein levels. (B) ∼19 kDa monomer alpha-synuclein levels of normal (NEynuclein levels of NE and OE neuroblastoma cells. �SN = alpha-synuclein prE cells (*p ≤ 0.05; **p ≤ 0.01; ***p < 0.001; n = 3; unpaired Student’s t-test).

as not toxic to either cell type after 1 h aggregation. In con-rast, NE and OE cells containing fibrillar NAC, resulting fromn 8-day pre-aggregation, exhibited 56 ± 4.8% and 32 ± 3.7%iability (Fig. 4).

.5. The effects of classical apoptotic factors on cell

iability

Three different classical apoptotic agents were used to exam-ne their effects on cell viability. After H2O2, amphotericin

ig. 4. The effects of the treatment with various aggregating state of A�(1-2) and NAC peptides on the two differentiated (NE and OE) cell lines asevealed by MTT assay. The initial cell number was 2 × 105/ml in both cellines. �SN = alpha-synuclein protein; A�(1-42) = amyloid beta 1-42 peptide;AC = non-amyloid component of Alzheimer’s disease amyloid; NE = alpha-

ynuclein normal expressing; OE = alpha-synuclein overexpressing. Differences*) compared with the non-treated control values (100%) and differences (#)ompared with the alpha-synuclein NE neuroblastoma cell values are signifi-ant at a level p ≤ 0.05, n = 21. Statistical analysis by ANOVA post hoc test,onferroni.

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overexpressing (OE) neuroblastoma cells. (C) ∼24–26 kDa monomer alpha-AU = arbitrary unit. Asterisks indicate significant difference between NE and

and ruthenium red treatment, the cell viability of NE cellsecreased to approximately 50% of control (IC50, n = 21). Thentreated control value was 100%. Strikingly, �SN OE cellsere less sensitive to cell damage induced by these agents

Fig. 5).

ig. 5. Differentiated NE and OE cells were treated by classical apoptotic fac-ors. Cell viability was measured by MTT assay. Alpha-synuclein OE cells wereess sensitive to cell damage induced by these agents, than the NE counter-arts. �SN = alpha-synuclein protein; NE = alpha-synuclein normal expressing;E = alpha-synuclein overexpressing; p.a. = pre-aggregated. Differences com-ared with the IC50 values are significant at a level *p ≤ 0.01, or at **p ≤ 0.001;= 21. Statistical analysis by ANOVA post hoc test, Bonferroni.

652 A. Hunya et al. / Brain Research Bulletin 75 (2008) 648–654

Fig. 6. The effects of A�(1-42) and NAC stressors (8-day aggregated form) on cell motility of the two differentiated (NE and OE) cell cultures. �SN = alpha-s ompoO ated cc = 30.

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ynuclein protein; A�(1-42) = amyloid beta 1-42 peptide; NAC = non-amyloid cE = alpha-synuclein overexpressing. Differences (*) compared with the non-tre

ell subjected to the same treatment; values are significant at a level p ≤ 0.01, n

.6. The effects of Aβ(1-42) and NAC treatment on cellotility

Cell motility of differentiated cells was measured after a0 min treatment period of aggregated (fibrillar form) A�(1-42)r NAC. Values were expressed as %, where 100% was the non-reated control value, and 0% corresponded to the fixed cells.lthough both aggregated peptides significantly inhibited theobility of neurites (52 ± 5.7% for A�(1-42) and 57 ± 5.2%

or NAC, n = 30, for NE cells); OE cells displayed a more ampleecrease of motility (35 ± 5.2% for A�(1-42) and 40 ± 4.0% forAC, n = 30) (Fig. 6).

. Discussion

A growing body of evidence indicates the central role of �SNn several neurodegenerative pathology, such as Parkinson’s andlzheimer’s disease. However, the exact mechanisms by whichSN contributes to the aetiology of cell death remain elusive.e have compared the viability of cells expressing normal (NE),

r elevated amount (OE) of �SN after various stressors. The ele-ated level of �SN was revealed by Western blot analysis. OEells were less vulnerable to classical apoptotic factors; however,ggregated, disease-related peptides induced greater damage tohis cell line, compared to NE. Furthermore, �SN overexpress-ng cells displayed altered phenotype, namely, they increasedeurite lengths and arborisation accompanied by decreased cellotility.Elevated level of �SN deteriorates microtubule-dependent

rafficking [24], which can result in the observed altered phe-otype [34]. In addition, the proliferation kinetics of OE cellsas much slower than that of NE cells. This might be due to

he inherent toxicity of the abnormal �SN level [21], or alterna-

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nent of Alzheimer’s disease amyloid; NE = alpha-synuclein normal expressing;ontrol values (100%) and differences (#) compared with the NE neuroblastomaStatistical analysis by ANOVA post hoc test, Bonferroni.

ively, OE cells with greater neurite arborisation simply do notivide as quickly, as the NE counterpart [41].

Several experimental data suggest that programmed celleath could contribute to Parkinson’s disease neuropathology30] and deficits in �SN regulation per se may elicit apoptosis21]. On the contrary, our data and some recent reports [8,26,40]how the anti-apoptotic or neuroprotective property of wild-typeSN overexpression. To explain this phenomenon, da Costa etl. [8] pointed out the possible chaperon-like property of �SN,s �SN exhibits a 40% homology with members of the 14-3-chaperone protein family [31]. Therefore, �SN may bind to

ellular mediators of the apoptotic pathways, counteracting theurther progress of programmed cell death. On the other hand,owever, �SN OE cells were more sensitive to aggregated A�(1-2) and NAC treatment than the NE counterparts. Presumably,ifferent apoptotic pathways become activated upon treatmentith aggregated peptides and apoptotic factors. A�(1-42) andAC may induce intracellular tau hyperphosphorylation andggregation [3,42], which subsequently facilitates the aggrega-ion of �SN [16], and eventually leads to the collapse of the

icrotubule system. Moreover, tau and �SN synergistically pro-ote and propagate the polymerization of each other into toxic

myloid fibrils [11,16], but each protein preferentially formsomopolymeric rather than heteropolymeric filaments [16]. Thisicious cycle can be more pronounced in the OE cells, whichb ovo contains an elevated level of �SN, therefore, A�(1-42)ay cause a more extensive cell damage. In support of this the-

ry, as we have shown earlier, the rate of damaging impact ofxogenous A�(1-42) on SH-SY5Y cells depends on the level of

rborisation [10]. Greater arborisation is accompanied by a moreeveloped microtubule system, and a higher level of tau protein,herefore exogenous aggregated peptide-triggered intracellularau and �SN may cause a more pronounced cellular damage.

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In contrast, the classical apoptotic factors used in this studyo not induce hyperphosphorylation and aggregation of tau orSN directly. Amphotericin B serves as a potassium permeableore-forming agent, which causes K+ leakage and subsequentpoptotic pathway activation [13,39]. Ruthenium red inhibitshe transport of calcium through membrane channels and impairs

itochondrial Ca2+-homeostasis [6], while H2O2 induces oxida-ive damage. On the contrary, aggregated peptides affect the

icrotubule system, rendering tau aggregation, which eventu-lly leads to �SN aggregation.

In conclusion, we have found that �SN overexpression pro-ects cells from classical apoptotic factors, while it renders cellsore vulnerable to aggregated A�(1-42) and NAC. This phe-

omenon may be exploited: the neuroprotective effect of novelompounds could be measured more precisely on OE cells owingo the higher sensitivity towards A�(1-42) and NAC. Thus,he potentially protective agents can be tested in a wider con-entration range. Furthermore, anti-neurodegenerative diseaseompounds could also be assayed in this sensitive cell model.inally, the �SN OE human SH-SY5Y neuroblastoma cell lineould serve as a good and effective in vitro model of both Parkin-on’s and Alzheimer’s diseases.

isclosure statement

All authors declare that they have no competing financial orersonal interests.

cknowledgement

Special thanks to Alex Liu, Department of Neurology, Uni-ersity of Saarland, Homburg, Germany for placing the two cellines at our disposal.

This work was supported by the National Bureau of Researchnd Development (NKTH RET 08/2004) and by grants of theational Research Foundation OTKA TS049817 and NKFPA/005/2004 OM-00424/2004 (MediChem 2).

eferences

[1] A. Abeliovich, Y. Schmitz, I. Farinas, D. Choi-Lundberg, W.H. Ho, P.E.Castillo, N. Shinsky, J.M. Verdugo, M. Armanini, A. Ryan, M. Hynes, H.Phillips, D. Sulzer, A. Rosenthal, Mice lacking alpha-synuclein displayfunctional deficits in the nigrostriatal dopamine system, Neuron 25 (2000)239–252.

[2] P. Ariano, C. Distasi, A. Gilardino, P. Zamburlin, M. Ferraro, A sim-ple method to study cellular migration, J. Neurosci. Methods 141 (2005)271–276.

[3] M. Blurton-Jones, F.M. Laferla, Pathways by which Abeta facilitates taupathology, Curr. Alzheimer Res. 3 (2006) 437–448.

[4] N.M. Bonini, B.I. Giasson, Snaring the function of alpha-synuclein, Cell123 (2005) 359–361.

[5] V. Charles, E. Mezey, P.H. Reddy, A. Dehejia, T.A. Young,M.H. Polymeropoulos, M.J. Brownstein, D.A. Tagle, Alpha-synucleinimmunoreactivity of huntingtin polyglutamine aggregates in striatum and

cortex of Huntington’s disease patients and transgenic mouse models, Neu-rosci. Lett. 289 (2000) 29–32.

[6] S.M. Cibulsky, W.A. Sather, Block by ruthenium red of cloned neuronalvoltage-gated calcium channels, J. Pharmacol. Exp. Ther. 289 (1999)1447–1453.

[

ulletin 75 (2008) 648–654 653

[7] N.B. Cole, D.D. Murphy, The cell biology of alpha-synuclein: a stickyproblem? Neuromol. Med. 1 (2002) 95–109.

[8] C.A. da Costa, K. Ancolio, F. Checler, Wild-type but not Parkinson’sdisease-related ala-53 → Thr mutant alpha-synuclein protects neuronalcells from apoptotic stimuli, J. Biol. Chem. 275 (2000) 24065–24069.

[9] Z. Datki, A. Juhasz, M. Galfi, K. Soos, R. Papp, D. Zadori, B. Penke,Method for measuring neurotoxicity of aggregating polypeptides with theMTT assay on differentiated neuroblastoma cells, Brain Res. Bull. 62(2003) 223–229.

10] Z. Datki, R. Papp, D. Zadori, K. Soos, L. Fulop, A. Juhasz, G. Laskay, C.Hetenyi, E. Mihalik, M. Zarandi, B. Penke, In vitro model of neurotoxicityof Abeta 1-42 and neuroprotection by a pentapeptide: irreversible eventsduring the first hour, Neurobiol. Dis. 17 (2004) 507–515.

11] T. Duka, M. Rusnak, R.E. Drolet, V. Duka, C. Wersinger, J.L. Goudreau, A.Sidhu, Alpha-synuclein induces hyperphosphorylation of Tau in the MPTPmodel of parkinsonism, FASEB J. 20 (2006) 2302–2312.

12] F. Flood, E. Sundstrom, E.B. Samuelsson, B. Wiehager, A. Seiger, J.A.Johnston, R.F. Cowburn, Presenilin expression during induced differentia-tion of the human neuroblastoma SH-SY5Y cell line, Neurochem. Int. 44(2004) 487–496.

13] G. Fujii, J.E. Chang, T. Coley, B. Steere, The formation of amphotericin Bion channels in lipid bilayers, Biochemistry 36 (1997) 4959–4968.

14] L. Fulop, M. Zarandi, Z. Datki, K. Soos, B. Penke, Beta-amyloid-derived pentapeptide RIIGLa inhibits Abeta(1-42) aggregation and toxicity,Biochem. Biophys. Res. Commun. 324 (2004) 64–69.

15] R.A. Furlong, Y. Narain, J. Rankin, A. Wyttenbach, D.C. Rubinsztein,alpha-synuclein overexpression promotes aggregation of mutant hunt-ingtin, Biochem. J. 346 (Pt 3) (2000) 577–581.

16] B.I. Giasson, M.S. Forman, M. Higuchi, L.I. Golbe, C.L. Graves, P.T.Kotzbauer, J.Q. Trojanowski, V.M. Lee, Initiation and synergistic fibril-lization of tau and alpha-synuclein, Science 300 (2003) 636–640.

17] M. Goedert, Alpha-synuclein and neurodegenerative diseases, Nat. Rev.Neurosci. 2 (2001) 492–501.

18] M. Hasegawa, H. Fujiwara, T. Nonaka, K. Wakabayashi, H. Takahashi,V.M. Lee, J.Q. Trojanowski, D. Mann, T. Iwatsubo, Phosphorylated alpha-synuclein is ubiquitinated in alpha-synucleinopathy lesions, J. Biol. Chem.277 (2002) 49071–49076.

19] A. Iwai, E. Masliah, M.P. Sundsmo, R. DeTeresa, M. Mallory, D.P. Salmon,T. Saitoh, The synaptic protein NACP is abnormally expressed duringthe progression of Alzheimer’s disease, Brain Res. 720 (1996) 230–234.

20] R. Kruger, A.M. Vieira-Saecker, W. Kuhn, D. Berg, T. Muller, N. Kuhnl,G.A. Fuchs, A. Storch, M. Hungs, D. Woitalla, H. Przuntek, J.T. Epplen, L.Schols, O. Riess, Increased susceptibility to sporadic Parkinson’s diseaseby a certain combined alpha-synuclein/apolipoprotein E genotype, Ann.Neurol. 45 (1999) 611–617.

21] B. Kumar, P. Nahreini, A.J. Hanson, C. Andreatta, J.E. Prasad, K.N. Prasad,Selenomethionine prevents degeneration induced by overexpression ofwild-type human alpha-synuclein during differentiation of neuroblastomacells, J. Am. Coll. Nutr. 24 (2005) 516–523.

22] U.K. Laemmli, Cleavage of structural proteins during the assembly of thehead of bacteriophage T4, Nature 227 (1970) 680–685.

23] M.P. Lambert, G. Stevens, S. Sabo, K. Barber, G. Wang, W. Wade, G.Krafft, S. Snyder, T.F. Holzman, W.L. Klein, Beta/A4-evoked degenerationof differentiated SH-SY5Y human neuroblastoma cells, J. Neurosci. Res.39 (1994) 377–385.

24] H.J. Lee, F. Khoshaghideh, S. Lee, S.J. Lee, Impairment of microtubule-dependent trafficking by overexpression of alpha-synuclein, Eur. J.Neurosci. 24 (2006) 3153–3162.

25] Y. Liu, D.A. Peterson, H. Kimura, D. Schubert, Mechanism of cellu-lar 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)reduction, J. Neurochem. 69 (1997) 581–593.

26] A.B. Manning-Bog, A.L. McCormack, M.G. Purisai, L.M. Bolin, D.A. Di

Monte, Alpha-synuclein overexpression protects against paraquat-inducedneurodegeneration, J. Neurosci. 23 (2003) 3095–3099.

27] L. Maroteaux, J.T. Campanelli, R.H. Scheller, Synuclein: a neuron-specificprotein localized to the nucleus and presynaptic nerve terminal, J. Neurosci.8 (1988) 2804–2815.

6 arch B

[

[

[

[

[

[

[

[

[

[

[

[

[

[

54 A. Hunya et al. / Brain Rese

28] W. Marui, E. Iseki, K. Ueda, K. Kosaka, Occurrence of human alpha-synuclein immunoreactive neurons with neurofibrillary tangle formationin the limbic areas of patients with Alzheimer’s disease, J. Neurol. Sci. 174(2000) 81–84.

29] E. Masliah, A. Iwai, M. Mallory, K. Ueda, T. Saitoh, Altered presynapticprotein NACP is associated with plaque formation and neurodegenerationin Alzheimer’s disease, Am. J. Pathol. 148 (1996) 201–210.

30] M.P. Mattson, Neuronal life-and-death signaling, apoptosis, and neurode-generative disorders, Antioxid. Redox Signal 8 (2006) 1997–2006.

31] N. Ostrerova, L. Petrucelli, M. Farrer, N. Mehta, P. Choi, J. Hardy, B.Wolozin, alpha-Synuclein shares physical and functional homology with14-3-3 proteins, J. Neurosci. 19 (1999) 5782–5791.

32] M.H. Polymeropoulos, C. Lavedan, E. Leroy, S.E. Ide, A. Dehejia, A.Dutra, B. Pike, H. Root, J. Rubenstein, R. Boyer, E.S. Stenroos, S. Chan-drasekharappa, A. Athanassiadou, T. Papapetropoulos, W.G. Johnson,A.M. Lazzarini, R.C. Duvoisin, G. Di Iorio, L.I. Golbe, R.L. Nussbaum,Mutation in the alpha-synuclein gene identified in families with Parkinson’sdisease, Science 276 (1997) 2045–2047.

33] S.P. Presgraves, T. Ahmed, S. Borwege, J.N. Joyce, Terminally differenti-ated SH-SY5Y cells provide a model system for studying neuroprotectiveeffects of dopamine agonists, Neurotox. Res. 5 (2004) 579–598.

34] Y. Rui, P. Tiwari, Z. Xie, J.Q. Zheng, Acute impairment of mitochondrialtrafficking by beta-amyloid peptides in hippocampal neurons, J. Neurosci.26 (2006) 10480–10487.

35] H. Shimura, M.G. Schlossmacher, N. Hattori, M.P. Frosch, A. Trocken-bacher, R. Schneider, Y. Mizuno, K.S. Kosik, D.J. Selkoe, Ubiquitination

[

ulletin 75 (2008) 648–654

of a new form of alpha-synuclein by parkin from human brain: implicationsfor Parkinson’s disease, Science 293 (2001) 263–269.

36] M.G. Spillantini, R.A. Crowther, R. Jakes, M. Hasegawa, M. Goedert,alpha-Synuclein in filamentous inclusions of Lewy bodies from Parkin-son’s disease and dementia with Lewy bodies, Proc. Natl. Acad. Sci. U.S.A.95 (1998) 6469–6473.

37] P.H. Tu, J.E. Galvin, M. Baba, B. Giasson, T. Tomita, S. Leight, S. Nakajo,T. Iwatsubo, J.Q. Trojanowski, V.M. Lee, Glial cytoplasmic inclusions inwhite matter oligodendrocytes of multiple system atrophy brains containinsoluble alpha-synuclein, Ann. Neurol. 44 (1998) 415–422.

38] K. Ueda, H. Fukushima, E. Masliah, Y. Xia, A. Iwai, M. Yoshimoto, D.A.Otero, J. Kondo, Y. Ihara, T. Saitoh, Molecular cloning of cDNA encodingan unrecognized component of amyloid in Alzheimer disease, Proc. Natl.Acad. Sci. U.S.A. 90 (1993) 11282–11286.

39] D.E. Varlam, M.M. Siddiq, L.A. Parton, H. Russmann, Apoptosis con-tributes to amphotericin B-induced nephrotoxicity, Antimicrob. AgentsChemother. 45 (2001) 679–685.

40] S. Vartiainen, V. Aarnio, M. Lakso, G. Wong, Increased lifespan in trans-genic Caenorhabditis elegans overexpressing human alpha-synuclein, Exp.Gerontol. 41 (2006) 871–876.

41] B. Winner, D.C. Lie, E. Rockenstein, R. Aigner, L. Aigner, E. Masliah, H.G.

Kuhn, J. Winkler, Human wild-type alpha-synuclein impairs neurogenesis,J. Neuropathol. Exp. Neurol. 63 (2004) 1155–1166.

42] W.H. Zheng, S. Bastianetto, F. Mennicken, W. Ma, S. Kar, Amyloid betapeptide induces tau phosphorylation and loss of cholinergic neurons in ratprimary septal cultures, Neuroscience 115 (2002) 201–211.