research article dopamine cytotoxicity involves both...

Research ArticleDopamine Cytotoxicity Involves Both Oxidative andNonoxidative Pathways in SH-SY5Y Cells Potential Role ofAlpha-Synuclein Overexpression and Proteasomal Inhibition inthe Etiopathogenesis of Parkinsonrsquos Disease

Kalpita Banerjee12 Soumyabrata Munshi13 Oishimaya Sen1 Vishmadeb Pramanik4

Tapasi Roy Mukherjee15 and Sasanka Chakrabarti1

1 Department of Biochemistry Institute of Post-Graduate Medical Education amp Research 244B Acharya J C Bose RoadKolkata 700020 India

2Department of Neuroscience Burke-Cornell Medical Research Institute 785 Mamaroneck Avenue White Plains NY 10605 USA3Department of Neuroscience Rosalind Franklin University of Medicine and Science 3333 Green Bay Road North ChicagoIL 60064 USA

4DNA Laboratory Anthropological Survey of India 27 J N Road Kolkata 700016 India5 National Institute of Cholera and Enteric Diseases P-33 CIT Road Kolkata 700010 India

Correspondence should be addressed to Sasanka Chakrabarti profschakrabarti95gmailcom

Received 6 November 2013 Revised 19 February 2014 Accepted 25 February 2014 Published 2 April 2014

Academic Editor Nobutaka Hattori

Copyright copy 2014 Kalpita Banerjee et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Background The cytotoxic effects of dopamine (DA) on several catecholaminergic cell lines involve DA oxidation products likereactive oxygen species (ROS) and toxic quinones and have implications in the pathogenesis of sporadic Parkinsonrsquos disease (PD)However many molecular details are yet to be elucidated and the possible nonoxidative mechanism of dopamine cytotoxicityhas not been studied in great detail Results Cultured SH-SY5Y cells treated with DA (up to 400 120583M) or lactacystin (5 120583M) orDA (400120583M) plus N-acetylcysteine (NAC 25mM) for 24 h are processed accordingly to observe the cell viability mitochondrialdysfunctions oxidative stress parameters proteasomal activity expression of alpha-synuclein gene and intracellular accumulationof the protein DA causesmitochondrial dysfunction and extensive loss of cell viability partially inhibited byNAC potent inhibitionof proteasomal activity marginally prevented by NAC and overexpression with accumulation of intracellular alpha-synucleinpartially preventable by NAC Under similar conditions of incubation NAC completely prevents enhanced production of ROS andincreased formation of quinoprotein adducts in DA-treated SH-SY5Y cells Separately proteasomal inhibitor lactacystin causesaccumulation of alpha-synuclein as well as mitochondrial dysfunction and cell death Conclusions DA cytotoxicity includes bothoxidative and nonoxidative modes and may involve overexpression and accumulation of alpha-synuclein as well as proteasomalinhibition

1 Introduction

Parkinsonrsquos disease (PD) which is a progressive neurodegen-erative disorder affecting mainly the elderly people appearsin two major forms familial and sporadic and the lattervariety accounts for nearly 90ndash95 of PD subjects Thepathological hallmark of PD is the degeneration of dopamin-ergic neurons of substantia nigra that project into striatum

[1] The underlying mechanisms of dopaminergic neuronaldeath in sporadic PD are still uncertain but mitochondrialdysfunctions oxidative stress proteolytic impairment withabnormal accumulation of proteins like alpha-synuclein andinflammatory reactions are key elements in this complexpathogenesis [1ndash3] Several animal and cell-based exper-imental models have been widely used to elucidate themechanism of dopaminergic neuronal death in sporadic

Hindawi Publishing CorporationParkinsonrsquos DiseaseVolume 2014 Article ID 878935 12 pageshttpdxdoiorg1011552014878935

2 Parkinsonrsquos Disease

PD [4ndash8] Although in some models various toxins like6-hydroxydopamine (6-OHDA) rotenone and 1-methyl-4-phenyl-1236-tetrahydropyridine (MPTP) have been used toinduce cell death the cytotoxic action of dopamine (DA)on catecholaminergic cell lines is of special relevance in thecontext of PD pathogenesis because DA is endogenouslyavailable in the body [5 6 9 10] A large body of evidencehas already implicated that DA oxidation products likereactive oxygen species (ROS) and toxic quinones can triggerapoptotic death in cultured PC12 and SH-SY5Y cells inwhich mitochondrial dysfunction plays a critical role [11ndash16] Although most of these studies on DA cytotoxicity havesuggested the oxidative mode of cellular damage a few havealso indicated the existence of a nonoxidative pathway in thisprocess [17 18]

The involvement of proteasomal impairment with abnor-mal accumulation of proteins especially alpha-synucleinin PD pathogenesis has been suggested based on multiplelines of evidence in sporadic and familial PD [19 20] Asubstantial body of evidence also indicates the toxic effectsof alpha-synuclein on different cell lines or on isolated brainmitochondria [21ndash26] Since proteasomal inactivation andalpha-synuclein accumulation occur in the substantia nigralneurons in PD it is likely that these phenomena have someunknown links with dopamine We hypothesize that alpha-synuclein accumulation and proteasomal inactivationmay belinked with either the oxidative or nonoxidative mode of DAtoxicity which could be tested in a cell-based model In thepresent study we have therefore exposed SH-SY5Y cells (aneuroblastoma cell line with the robust expression of alpha-synuclein) to varying concentrations of DA over a periodof 24 h with or without radical and quinone scavengers andmeasured cytotoxicity parameters as well as alpha-synucleincontent and proteasomal activity

2 Materials

The reagents used in the studywere obtained from the follow-ing sources DulbeccorsquosModified EaglersquosMediumNutrient F-12Ham (DMEMF-12Ham 1 1) fetal bovine serum (FBS)551015840661015840-tetrachloro-111015840331015840-tetraethylbenzimidazolocarb-ocyanine iodide (JC-1) ATP bioluminescent assay kit peni-cillin streptomycin and amphotericin B fromSigmaAldrichUSA dopamine (DA) N-acetylcysteine (NAC) and AmplexRed from Acros Organics USA fluorogenic peptides (Suc-Leu-Leu-Val-Tyr-AMC and Z-Leu-Leu-Glu-AMC) and lacta-cystin from Alexis Biochemicals USA nitroblue tetrazolium(NBT) from Promega India polyclonal rabbit anti-alpha-synuclein antibody from Santa Cruz Biotechnology USAhorseradish peroxidase- (HRP-) conjugated secondary goatanti-rabbit IgG antibody from Bangalore Genei India RNAextraction kit from Roche Applied Science Germany andreverse transcription kit forward and reverse primers forboth the target (alpha-synuclein) and the reference (120573-actin) genes from Roche Diagnostics Germany All commonchemicals and reagents used were of the highest analyticalgrade available and were obtained from Sisco ResearchLaboratories India

3 Methods

31 Cell Culture and Treatment Paradigm Undifferentiatedhuman neuroblastoma SH-SY5Y cells obtained from Amer-ican Type Culture Collection (ATCC USA) were grownin DMEMF-12Ham containing 10 (vv) heat-inactivatedFBS 50 unitsmL penicillin 50 120583gmL streptomycin and25 120583gmL amphotericin B in a humidified environmentcontaining 5 CO

2and 95 air at 37∘C in 25 cm2 sterile

tissue culture flasks SH-SY5Y cells (70ndash75 confluent) wereincubated with or without varying concentrations of DA(100ndash400 120583M) in the absence or presence of other additionslike NAC (25mM) or lactacystin (5 120583M) for 24 h Controland treated cells were analyzed for cell death mitochondrialfunctions oxidative stress parameters proteasomal activ-ity and alpha-synuclein content and alpha-synuclein geneexpression

32 Assessment of Cell Death

321 Trypan Blue Assay An aliquot of cell suspension inphosphate-buffered saline (PBS) was mixed with an equalvolume of trypan blue solution (04 in PBS) and the cellswere counted in a Neubauer chamber The cell death wasexpressed as the percentage of trypan blue positive cells in thetotal population of stained and unstained cells counted by anobserver blind to treatment protocol [27]

322 Lactate Dehydrogenase (LDH) Assay Cell death wasalso measured by LDH assay in which the activity of LDHreleased in the medium expressed as the percentage of thetotal LDH (intracellular plus extracellular) was measuredusing NADH-based spectrophotometric assay [28]

33 Measurement of Mitochondrial Membrane Potential inSH-SY5YCells Mitochondrial transmembrane potential wasmeasured in intact SH-SY5Y cells by using a cationic carbo-cyanine dye JC-1 which remains distributed in the cytosolas monomers while after entering the mitochondria drivenby the electrochemical gradient undergoes concentration-dependent aggregation to form J-aggregates The monomersemit a green fluorescence (120582ex 490 nm 120582em 530 nm) and J-aggregates a red fluorescence (120582ex 490 nm 120582em 590 nm) andthe ratio of fluorescence intensities at 590 nm to 530 nm indi-cates mitochondrial transmembrane potential [11] Brieflycontrol and treated cells were washed twice in PBS andthen incubated in serum-free DMEM for 30min in thepresence of JC-1 (10 120583M) at 37∘C in the dark The cellswere pelleted down and then washed thrice with serum-free DMEM suspended with suitable dilutions in the samemedium and the fluorescence emission intensities (590 nmand 530 nm) were measured with excitation at 490 nm ina spectrofluorometer (FP-6300 JASCO International CoJapan)

34 Measurement of ATP Content in SH-SY5Y Cells TheATP content was measured in control and treated SH-SY5Y cells by using a commercial kit based on luciferin-luciferase reaction as described previously [11] Briefly the

Parkinsonrsquos Disease 3

treated and control cells were washed with PBS followed bydisruption of resuspended cells in 100 120583L aliquots by ice-cold lysis-buffer containing 10mM Tris 1mM EDTA and05 Triton X-100 pH 76 They were immediately used forATP measurement using the ATP-assay mix provided by thekit [11] Using a microplate luminometer (Biotek ELX-800USA) the luminescence generated was measured promptlyAppropriate blank reactions consisting of ATP-assay mixand lysis buffer without cells were performed to measurebackground luminescence The intracellular content of ATPwas calculated from an ATP standard curve generated byplotting the luminescence readings against known concen-trations of ATP (008 nmolmLndash40 nmolmL) and the valueswere calculated as nmol of ATP per mg of protein

35 Assessment of Oxidative Stress Parameters inSH-SY5Y Cells

351 Measurement of Hydrogen Peroxide (H2O2) Genera-

tion Using Amplex Red H2O2production from SH-SY5Y

cells during in vitro incubation was measured using theH2O2-specific fluorescent dye Amplex Red (N-acetyl-37-

dihydroxyphenoxazine) as described earlier [11 29] Brieflycontrol and treated cells were harvested and washed twicewith phosphate-buffered saline (PBS) and finally resus-pended in Krebs-Ringerrsquos buffer containing 10mM glucose(pH 74) An aliquot of this cell suspension was incubatedin the same Krebs-Ringerrsquos buffer with 50120583M Amplex Redand 1UmL horseradish peroxidase for 15min in the dark atroom temperature At the end of the incubation an aliquotof the incubation mixture was appropriately diluted andthe fluorescence emission was measured at an excitationwavelength of 530 nm and an emission wavelength of 590 nmusing a spectrofluorometer (FP6300 JASCO InternationalCo Japan) The background fluorescence was subtractedfrom the sample fluorescence using a blank containingthe reaction mixture without any added cells Fluorescenceintensity was converted to nmol of H

2O2produced per mg of

protein using a calibration curve utilizing pure H2O2in the

concentration range of 50 nM to 400 nM

352 Nitroblue Tetrazolium (NBT)Glycinate Assay Quino-protein adduct formation in SH-SY5Y cells was measured bythe NBTglycinate assay following published procedures [3031] Briefly control and treated cells were first washed withphosphate-buffered saline and then suspended in 50mMphosphate buffer (pH 74) in a total volume of 200 120583Lfollowed by addition of 200 120583L of 10 trichloroacetic acidfor protein precipitation The precipitate was washed twicewith ethanol treated with chloroform-methanol (2 1 vv)and vortexed thoroughly It was then centrifuged at 5000 g for10min and the supernatant was removed To the delipidatedprotein precipitate 1mL of NBT reagent (024mM NBT in2M potassium glycinate pH 10) was added followed byincubation in the dark for 1 h on a shaker The blue-purplecolor developed in the reaction mixture was suitably dilutedand absorbance taken at 530 nm using a spectrophotometer(model 117 Systronics India)

36 Proteasomal Degradation Assay Proteasome activity wasassayed in the cell lysate bymeasuring the release of 7-amino-4-methylcoumarin (AMC) from the fluorogenic peptidesSuc-Leu-Leu-Val-Tyr-AMC and Z-Leu-Leu-Glu-AMC Cellswere lysed in 20mM Tris-HCl 1mM EDTA buffer pH 75by 4 cycles of freezing-thawing followed by centrifugation at5000 rpm for 10min The supernatant was collected and analiquot (100ndash300 120583g protein) was added to the assay mixturein a total volume of 300 120583L containing 50120583M Z-Leu-Leu-Glu-AMC 50120583M Suc-Leu-Leu-Val-Tyr-AMC 5mM ATPand 5mM Mg acetate in Tris-EDTA buffer with or withoutlactacystin (01 120583M) The assay mix was incubated at 37∘Cfor 30min followed by the fluorescence measurement at 120582ex360 nm and 120582em 465 nm The assay produced proportionalresponse up to 300 120583g of protein Lactacystin sensitive activitywas taken as the proteasomal activity and expressed asarbitrary fluorescence units100120583g of protein [20]

37 Immunodetection of Alpha-Synuclein in SH-SY5Y CellsSH-SY5Y cells were lysed in cell lysis buffer (20mM Tris1mM EDTA pH 74) by repeated freeze-thaw The lysatewas then centrifuged for 10min at 12000 rpm at 4∘CThe supernatant was subjected to 12 SDS-PAGE followedby immunoblotting utilizing standard blotting protocolsand using primary antibody (polyclonal rabbit anti-alpha-synuclein antibody 1 1000 dilution) and HRP-conjugatedsecondary goat anti-rabbit IgG antibody (1 5000 dilution)Finally the blot was developed by the enhanced chemilu-minescence technique Equal amount of protein was loadedin each well for SDS-PAGE The band intensities were mea-sured using a gel doc apparatus (G-Box Syngene UK) andanalyzed with the help of the software Syngene Gene Tools(File version 40104) using gamma-actin loading controlnormalization

38 Quantitative RT-PCR for Alpha-Synuclein GeneExpression Total RNA was extracted from control andtreated cells using a commercial kit (Roche AppliedScience Germany) and following the manufacturerrsquosprotocol Reverse transcription was performed as per themanufacturerrsquos protocol (Roche Diagnostics Germany)from total RNA utilizing random primers The real-time PCR analysis of the cDNA samples for a fragment(187 bp) of alpha-synuclein gene was performed in triplicateusing SYBR Green in a reaction volume of 20120583L using10 pmol each of forward and reverse primers for both thetarget (alpha-synuclein) and the reference 120573-actin genes(Applied Biosystems model 7500) The following primerswere used 120573-actin forwardmdashtcaccatggatgatgatatcgcc 120573-actin reversemdashccacacgcagctcattgtagaagg alpha-synucleinforwardmdashaggactttcaaaggccaagg and alpha-synucleinreversemdashtcctccaacatttgtcacttgc [32] The changes in alpha-synuclein gene expression in different samples werecalculated from the Ct (threshold cycle) values of the targetgene and the reference gene by the relative quantitationmethod [32]

39 Protein Estimation Theproteinwas estimated by Lowryrsquosmethod after solubilizing the samples in 1 SDS [33]


0

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ell d

eath

(per

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age o

f try

pan

blue

pos

itive

cells

)lowast

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Control DA(200120583M)

DA(400120583M)

DA(100120583M)

DA(400120583M)+ NAC

(a)

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ased

()

lowast

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DA(400120583M)

DA(100120583M)

DA(400120583M)+ NAC

(b)

Figure 1 Dose-dependent effects of DA on SH-SY5Y cell-viability SH-SY5Y cells were treated without (control) or with varyingconcentrations of DA (100ndash400120583M) for 24 h in the presence or absence of NAC (25mM) and the cell viability was measured by (a) trypanblue assay or (b) estimation of LDH activity as described in Section 3 Values are the means plusmn SD of six observations Statistically significantdifferences in cell death among the groups exist at 119875 lt 0001 as shown by asterisks (lowast) (in (a) 119865 (4 25) = 117709 119875 lt 0001 in (b) 119865 (4 25)= 89899 119875 lt 0001)

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JC-1

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ence

(590

nm5

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ratio

)

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(a)

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acel

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r ATP

cont

ent (

perc

enta

ge o

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lowast

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DA(400120583M)+ NAC

(b)

Figure 2 Effect of DA on mitochondrial functions in SH-SY5Y cells SH-SY5Y cells were incubated without or with DA (400 120583M) for 24 hin the absence or presence of NAC (25mM) and (a) mitochondrial membrane potential and (b) intracellular ATP content were measured asdescribed in Section 3 Values are the means plusmn SD of six observations Statistically significant differences (119875 lt 0001) exist among the groupsas shown by asterisks (lowast) (in (a) 119865 (2 15) = 35356 119875 lt 0001 in (b) 119865 (2 15) = 6739 119875 lt 0001)


0

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350

nmol

of h

ydro

gen

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xide

per

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of p

rote

in

lowastlowast


DA(400120583M)+ NAC(25 mM)

(a)

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ein

boun

d qu

inon

es (O

D 5

30m

g pr

otei

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lowast lowast


DA(400120583M)+ NAC(25 mM)

(b)

Figure 3Measurement of oxidative stress parameters (a) Effect ofNAC (25mM)onDA (400 120583M)-induced production of hydrogenperoxide(H2O2) in SH-SY5Y cells production of H

2O2was measured during in vitro incubation of SH-SY5Y cells (control 400120583M DA treated and

400 120583MDA + 25mMNAC treated) in Krebs-Ringerrsquos buffer as detailed in Section 3 Values (expressed as nmol of H2O2per mg protein) are

the means plusmn SD of four observations Statistically significant differences exist (119865 (2 9) = 13263 119875 lt 0001)) between the DA-treated groupsand control as well as between the DA-treated groups and NAC-treated groups as shown by asterisks (lowast) (b) Measurement of protein boundquinones in SH-SY5Y cells levels of protein bound quinones were measured in SH-SY5Y cells incubated without or with DA (400120583M) for24 h in the presence or absence of NAC (25mM) as detailed in Section 3 Values (optical density at 530 nm (OD 530) per mg protein) are themeans plusmn SD of four observations Statistically significant differences exist (119865 (2 9) = 3579 119875 lt 0001) between the DA-treated groups andcontrol as well as between the DA-treated groups and NAC-treated groups as shown by asterisks (lowast)

310 Statistical Analysis The statistical analysis of differentparameters among 3 or more groups was performed byanalysis of variance (ANOVA) followed by post hoc com-parison by means of Newman-Keuls test when the 119875 valuewas significant For the densitometric analysis Tukeyrsquos posthoc test was done The statistical comparison of differentparameters between 2 groups was performed by Studentrsquos119905-test paired The 119875 value less than 005 was consideredsignificant Each experiment was performed several timesand the values were expressed as the mean plusmn SD (standarddeviation) of the number of observations [34]

4 Results

41 DA Effects on Viability of SH-SY5Y Cells When SH-SY5Y cells were exposed to DA a dose-dependent loss ofcell viability was noted as measured by trypan blue exclusionassay (Figure 1(a)) The release of the cytosolic enzyme LDHin the medium reflecting cell death also increased signifi-cantly and in a dose-dependent manner after DA exposureof SH-SY5Y cells (Figure 1(b)) After 24 h of exposure to400 120583MDA a significant loss of cell viabilitywas noted in SH-SY5Y cells which was prevented to the extent of nearly 50by concomitant presence of N-acetylcysteine (25mM) in themedium (Figures 1(a) and 1(b)) N-acetylcysteine (25mM)

alone did not cause any significant effect on the cell viabilitycompared to control (data not shown)

42 Effect of DA onMitochondrial Function in SH-SY5Y CellsDA (400 120583M) for 24 h caused a striking loss of mitochondrialmembrane potential in SH-SY5Y cells as evident from asignificant (nearly 70) decrease in the ratio of fluorescenceintensities of JC-1 at 590 nm and 527 nm (Figure 2(a)) DAtreatment for the same period also decreased theATP contentof SH-SY5Y cells by nearly 40 as compared to control(Figure 2(b)) Both phenomena were prevented partially butsignificantly by concomitant presence of N-acetylcysteine(25mM) in the medium

43 Oxidative Stress Parameters in SH-SY5Y Cells after DA(400 120583M) Exposure Following DA (400120583M) exposure for24 h a significant increase of ROS production was observedin SH-SY5Y cells but the phenomenon was completelyprevented in cells co-treated with DA (400 120583M) and NAC(25mM) (Figure 3(a))

DA (400120583M)-derived quinones reacted with cellularnucleophiles to produce stable quinoprotein adducts AfterDA (400 120583M) treatment for 24 h the quinoprotein adductformation was increased by twofolds in SH-SY5Y cells which


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otea

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(per

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age o

f con

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DA(400120583M)+ NAC

lowast

lowast

lowast

Figure 4 Effect of DA on proteasomal activity in SH-SY5Y cellsProteasomal activity was measured in SH-SY5Y cells incubatedwithout or with DA (400120583M) for 24 h in the presence or absenceof NAC (25mM) as detailed in Section 3 Values (expressed aspercentage of the control) are the means plusmn SD of five observationsStatistically significant differences exist (119865 (2 12) = 35638 119875 lt0001) between the groups as shown by asterisks (lowast)

was completely reversed by concomitant treatment withNAC(25mM) (Figure 3(b))

44 Proteasomal Inhibition in SH-SY5Y Cells DA and Lac-tacystin Effects Proteasomal activity in SH-SY5Y cells wasdecreased by approximately 65 compared to that of controlafter exposure to DA (400 120583M) for 24 h and in the presenceof NAC (25mM) in the culture medium DA still producednearly 50 inhibition of the enzyme activity (Figure 4)

When SH-SY5Y cells were incubated with the protea-somal inhibitor lactacystin (5 120583M) for 24 h cell viabilitymeasured by trypan blue assay and released LDH activitywas significantly reduced (Figures 5(a) and 5(b)) Lactacystin(5 120583M) for 24 h also caused a striking loss of mitochondrialmembrane potential in SH-SY5Y cells as evident from asignificant (nearly 40) decrease in the ratio of fluorescenceintensities of JC-1 at 590 nm and 527 nm (Figure 6(a)) Fur-ther lactacystin (5 120583M) treatment for 24 h decreased the ATPcontent of SH-SY5Y cells by nearly 25 as compared tocontrol (Figure 6(b))

45 Alpha-Synuclein Accumulation in SH-SY5Y Cells afterExposure to DA or Lactacystin When SH-SY5Y cells wereexposed to DA (400 120583M) or lactacystin (5 120583M) for 24 h amarked increase in alpha-synuclein content was observedin either case compared to control using the Westernimmunoblotting technique and densitometric analysis (Fig-ures 7(a) and 7(b)) When the cells were co-treated with DA

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(a)

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ased

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lowast

(b)

Figure 5 Effect of lactacystin on SH-SY5Y cell-viability SH-SY5Ycells were treated without (control) or with lactacystin (5120583M) for24 h followed by the measurement of cell viability by (a) trypan bluemethod and (b) activity of released LDH as described in Section 3Values are the means plusmn SD of six observations Studentrsquos 119905-testpaired shows significant difference (119875 lt 0001) in the cell viabilitybetween the treated group (M = 3049 SD = 269) and the controlgroup (M = 401 SD = 172) 119905 (5) = 2423 as shown by asterisks (lowast)

(400 120583M) and NAC (25mM) for 24 h DA-induced increasein alpha-synuclein content was significantly prevented (Fig-ures 7(a) and 7(b))

46 Alpha-Synuclein Gene Expression in SH-SY5Y Cells afterExposure to DA The expression of alpha-synuclein gene inSH-SY5Y cells after exposure to DA (400 120583M) for 24 h wasincreased by 27-folds as measured by quantitative RT-PCRwhile only 15-fold increase was seen when the cells weretreated with DA (400 120583M) in the presence of 25mM NACfor the same period (Figure 8)

5 Discussion

DA- or L-34-dihydroxyphenylalanine- (L-DOPA-) inducedcell death has been widely studied in several catecholamin-ergic cell lines or primary neuronal culture from embryonicbrain and these are considered to be useful models tostudy PD pathogenesis [14 18 35 36] Both ROS and toxicquinones have been implicated in DA-induced cell death insuch models which can be effectively prevented by NACascorbate and other antioxidants suggesting an underlyingoxidative mechanism of DA toxicity [14 35ndash37] In a recentlypublished paper we have also shown that DA induces celldeath and mitochondrial dysfunctions in PC12 cells whichare mediated by DA-derived quinones and the phenomenonis nearly completely prevented by NAC which apart frombeing an antioxidant is also a potent scavenger of toxic


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(b)

Figure 6 Lactacystin effects on mitochondrial functions in SH-SY5Y cells SH-SY5Y cells were exposed to lactacystin (5 120583M) for24 h followed by the measurement of (a) mitochondrial mem-brane potential and (b) intracellular ATP content as described inSection 3 Values are the means plusmn SD of six observations Studentrsquos119905-test paired shows significant difference (lowast119875 lt 0001) in themitochondrial membrane potential between the lactacystin-treatedgroup (M = 839 SD = 071) and the control group (M = 1325 SD =122) 119905(5) = 1281 (a) Similarly a significant difference ( lowast119875 lt 001) inthe ATP content between the lactacystin-treated group (M = 7179SD = 1008) and the control group exists 119905 (5) = 379 (b)

quinones [11] In the present study a dose-dependent deathof cultured SH-SY5Y cells with dopamine is observed alongwith a significant mitochondrial functional impairmentwhich surprisingly enough are only partially preventableby NAC (25mM) (Figures 1 and 2) This concentrationof NAC however completely prevents the enhanced ROSproduction and increased quinoprotein adduct formationin DA-treated SH-SY5Y cells (Figures 3(a) and 3(b)) Weused more than sixfold higher concentration of NAC ascompared to DA to completely neutralize DA-derived ROSand toxic quinones Apart from directly scavenging ROSand active quinones NAC can also upregulate intracellularglutathione (GSH) adding to its antioxidant efficacyThus theresults (Figures 3(a) and 3(b)) suggest that DA cytotoxicityunder our experimental conditions is mediated partiallyby nonoxidative mechanisms which is in contrast to ourfindings in PC12 cells [11]

51 Proteasomal Inhibition and DA Cytotoxicity In ourpresent study we have tried to explore if the complexmechanism(s) underlying DA cytotoxicity could be relatedto DA-induced proteasomal inhibition in SH-SY5Y cells Itis observed that the proteasomal inhibition is only mildlyattenuated by treatment with a known antioxidant andquinone scavenger NAC (Figure 4) suggesting a primarynonoxidative mechanism of inhibition caused by DA At this

stage we hypothesized that proteasomal inhibition (causedby DA) accounts for the nonoxidative cytotoxic actions ofDA such as mitochondrial depolarization and cell deathobserved during co-treatment with the antioxidant quinonescavenger NAC (Figures 1 and 2) In support of the possi-bility that proteasomal inhibition may lead to mitochondrialdysfunction and cell death we have used a ldquoproteasomal-inhibition modelrdquo using a well-known proteasomal inhibitorlactacystin to examine the effects on the viability and mito-chondrial functions of SH-SY5Y cells Our results confirmthat proteasomal inhibition indeed causes a marked loss ofcell viability with mitochondrial membrane depolarizationand a decreased intracellular ATP content (Figures 5 and 6)thus validating our hypothesis

TheDA-induced proteasomal inhibition observed in thisstudy appears quite unusual because of the failure of NACa strong antioxidant and quinone scavenger to prevent theprocess In many earlier studies the oxidation products ofDA such as toxic quinones have been shown to inhibit severalenzymes like tyrosine hydroxylase tryptophan hydroxylaseand Na+K+ ATPase either in purified preparation or celllysate or crude brain membrane fractions [38ndash40] Under thepresent experimental conditions a direct effect of DA down-regulating the expression of proteasomal subunits appears asa distinct possibility

52 Alpha-Synuclein in Cytotoxicity of DA An earlier pub-lished study from our laboratory with isolated rat brainmitochondria has shown that alpha-synuclein interferes withmitochondrial bioenergetic functions which have implica-tions in pathogenesis of PD [25] Hence in our present studywe have attempted to find a possible link of DA cytotox-icity with alpha-synuclein Interestingly enough we havenoticed that DA causes an increase in the alpha-synucleincontent within SH-SY5Y cells (Figure 7) by enhancing geneexpression (Figure 8) and presumably also by inhibitingproteasomal degradation of alpha-synuclein (Figure 4) Thefact that lactacystin treatment of SH-SY5Y cells leads tothe intracellular accumulation of alpha-synuclein (Figure 7)confirms that the proteasomal degradation is an impor-tant mechanism of alpha-synuclein clearance It is furtherobserved that DA-induced alpha-synuclein overexpressionis only partially prevented by NAC whereas proteasomalinhibition is prevented even to a lesser extent (Figure 7)As a consequence when SH-SY5Y cells are exposed to DAa significant accumulation of alpha-synuclein occurs evenin the presence of NAC (Figure 7) The accumulated alpha-synuclein in DA and NAC co-treated cells could contributeto mitochondrial dysfunctions and eventual cell death andthismechanismmay be considered as a possible nonoxidativepathway of DA cytotoxicity In the absence of NAC co-treatment alpha-synuclein expression and accumulation iseven more pronounced (Figures 7 and 8) and would con-tribute to DA cytotoxicity in a larger measure

The underlying mechanism of alpha-synuclein overex-pression by DA is not evident from this study except for thefact that the phenomenon is partlymediated byDA oxidationproducts and partly by some nonoxidative pathwayHoweverother studies have indicated that DA causes enhancement


DA DA + NACControl Lactacystin

120572-Synuclein

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(a)

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-syn

ucle

in p

rote

in ex

pres

sion

(au

) lowast

lowast

lowastlowast

(b)

Figure 7 Accumulation of 120572-synuclein in SH-SY5Y cells after DA and lactacystin exposure SH-SY5Y cells were incubated alone (control)with DA (400 120583M) with lactacystin (5 120583M) and with 400 120583MDA co-treated with 25mMNAC for 24 h After 24 h of incubation cell lysateswere prepared and processed for Western blotting as described in Section 3 The specificity of the commercially available antibody has beenchecked against pure recombinant human 120572-synuclein by Western blotting and immunodot blotting (data not shown) (a) The immunoblotis representative of a set of identical experiments repeated four times (b) Densitometric analysis of the 120572-synuclein bands obtained by theWestern blot (lowast denotes 119875 lt 0001 between the groups)

0

05

1

15

2

25

3

35

4

45

Fold

incr

ease

of120572

-syn

ucle

in g

ene e

xpre

ssio

n

DA(400120583M)

DA(400120583M)+ NAC

lowast

Figure 8 DA effects on 120572-synuclein gene expression in SH-SY5Ycells SH-SY5Y cells were incubated alone (control) or with DA(400120583M) or 400 120583MDA plus 25mM NAC for 24 h Cellular RNAwas isolated and quantitative RT-PCR was carried out for 120572-synuclein gene as detailed in Section 3 Results expressed as thefold increase with respect to the control are the means plusmn SD ofsix observations Statistical significance collected by Studentrsquos 119905-testpaired shows significant difference (lowast119875 lt 005) in the fold of alpha-synuclein gene expression between the DA-treated group (M = 273SD = 109) and the NAC co-treated group (M = 157 SD = 034) 119905 (5)= 305

of alpha-synuclein gene expression by involving stress acti-vated protein kinase JNK and p38 or by upregulating theexpression of CEBP beta an enhancer-binding protein thatregulates the expression of alpha-synuclein [41] On the otherhand another study has suggested that DA by decreasing themethylation of CpG islands causes an upregulation of alpha-synuclein gene in several cell lines including SH-SY5Y andHEK293 cells [42]

53 Cytotoxicity of Alpha-Synuclein Corroboration withEarlier Studies Our present results implying that alpha-synuclein accumulation could be toxic to SH-SY5Y cells arein agreement with other studies where exogenously addedalpha-synuclein or overexpression of alpha-synuclein hasled to both mitochondrial impairment and cell death invarious cultured cell lines [23 42 43] Further anotherstudy has shown that downregulation of alpha-synuclein geneexpression occurs in HEK293 cells through adeno-associatedvirus (AAV) mediated delivery of alpha-synuclein ribozyme(SynRz) and the injection of the same AAV vector carryingSynRz in to substantia nigra of 1-methyl-4-phenylpyridinium(MPP+) treated rats prevents the apoptotic death of the nigraldopaminergic cells implying the toxic potential of elevatedintracellular alpha-synuclein [44] As previously mentionedthe toxic potential of alpha-synuclein on isolated rat brainmitochondria has also been shown in an earlier work in ourlaboratory [25] However there are scattered reports whichhave indicated a protective action of alpha-synuclein againstoxidative injury in neural cells [45 46] The dominant viewhowever tends to support the cytotoxic potential of alpha-synuclein [47]


Cell death

Mitochondrial dysfunctions

Dopaminecytotoxicity

Nonoxidative mechanism(not preventable by NAC)

Oxidative mechanism(preventable by NAC)

(a)

Dopamine exposure to

cells

Proteasomal inhibition





uarr

darr

degradationuarr

accumulation

gene expression

120572-Synuclein 120572-Synuclein

120572-Synuclein

(b)

Proteasomal inhibition model

Proteasomalinhibition

Cell death


Lactacystin

uarr 120572-Synuclein accumulation

(c)

Figure 9 Probable mechanisms of dopamine cytotoxicity in SH-SY5Y cells involvement of alpha-synuclein and proteasomal inhibition (a)shows that dopamine causes cell death and mitochondrial dysfunctions involving both the oxidative (NAC responsive) and nonoxidative(NAC nonresponsive) mechanisms (b) shows that dopamine causes proteasomal inhibition and increased alpha-synuclein gene expressioninvolving similar kind of oxidative (NAC responsive) and nonoxidative (NAC nonresponsive) mechanisms Both of these effects causeincreased intracellular accumulation of alpha-synuclein (c) shows our ldquoproteasomal inhibition modelrdquo using lactacystin resulting in celldeath mitochondrial dysfunctions and increased intracellular alpha-synuclein accumulation

6 Conclusions

Our study has highlighted some important aspects of DAcytotoxicity especially the nonoxidative mode in a cell-based model and suggested that proteasomal inactivationand alpha-synuclein accumulation are potential contributors

to this process Figure 9 provides a schematic diagram of theunderlying mechanisms involved in dopamine cytotoxicityas evident from this paper It remains unclear if thesephenomena are relevant in the actual pathogenesis of PDbut several findings are quite suggestive in this regardFor example a crucial evidence is the postmortem finding


showing proteasomal inactivation in substantia nigra of PDpatients with selective loss of 2026S proteasomal subunits[19] Further decreased methylation of intron 1 of alpha-synuclein gene leading to overexpression of this gene hasbeen reported in substantia nigra of postmortem brain of PDpatients [42] The alpha-synuclein content of normal brainis around 1 120583M while in postmortem PD brain the levelreported is much higher [48ndash50] Moreover alpha-synucleinis the predominant protein component of Lewy bodies whichappear in the degenerating nigral neurons in the brain of PDpatients [3 51] The genome-wide association studies havealso implicated alpha-synuclein gene with PD pathogenesis[52] All these facts emphasize the need for a more detailedstudy on proteasomal inactivation alpha-synuclein overex-pression and accumulation and toxicity in dopaminergicneuronal death in PD and DA-based cytotoxicity modelas used here could be very important for further elaboratestudies

Abbreviations

6-OHDA 6-HydroxydopamineAMC 7-Amino-4-methylcoumarinDA DopamineDMEMF-12 Ham Dulbeccorsquos Modified Eaglersquos

MediumNutrient F-12 HamFBS Fetal bovine serumHRP Horseradish peroxidaseJC-1 551015840661015840-Tetrachloro-111015840331015840-

tetraethylbenzimidazolocarbocyanineiodide

JNK c-Jun amino-terminal kinasesL-DOPA L-34-DihydroxyphenylalanineMPP+ 1-Methyl-4-phenylpyridiniumMPTP 1-Methyl-4-phenyl-1236-

tetrahydropyridineNAC N-AcetylcysteineNBT Nitroblue tetrazoliumPD Parkinsonrsquos diseaseROS Reactive oxygen species

Conflict of Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contribution

Kalpita Banerjee Soumyabrata Munshi and SasankaChakrabarti made substantial contributions to theconception and design of the study analysis of the data andwriting of the paper Kalpita Banerjee and Oishimaya Senperformed the cell viability and ATP assays Kalpita BanerjeeSoumyabrata Munshi and Oishimaya Sen did the Westernblot experiments and those involving the determination ofmitochondrial membrane potentials Vishmadeb Pramanikand Kalpita Banerjee conducted the quantitative real-timePCR experiments Tapasi Roy Mukherjee and KalpitaBanerjee performed the proteasomal assays Oishimaya Sen

performed the densitometric analysis of the immunoblotsand performed the assessment of oxidative stress parametersOishimaya Sen Tapasi RoyMukherjee Kalpita Banerjee andSoumyabrata Munshi were involved in maintenance of thecell lines Sasanka Chakrabarti managed the project checkedthe protocols and methods routinely and critically assessedthe data All authors read and approved of the final paperAll authors fulfilled the criteria for authorship SoumyabrataMunshi and Oishimaya Sen contributed equally to this work

Acknowledgments

This work was supported by the Board of Research inNuclearSciences Department of Atomic Energy Government ofIndia Mumbai India (no 20083728BRNS2369) KalpitaBanerjee was supported by an Award of Senior ResearchFellowship from the Indian Council of Medical ResearchNew Delhi India Oishimaya Sen was supported by SeniorResearch Fellowship from the Department of BiotechnologyNew Delhi India

References

[1] C W Olanow and W G Tatton ldquoEtiology and pathogenesis ofParkinsonrsquos diseaserdquoAnnual Review of Neuroscience vol 22 pp123ndash144 1999

[2] P M Abou-Sleiman M M K Muqit and N W WoodldquoExpanding insights of mitochondrial dysfunction in Parkin-sonrsquos diseaserdquoNature ReviewsNeuroscience vol 7 no 3 pp 207ndash219 2006

[3] T M Dawson and V L Dawson ldquoMolecular pathways ofneurodegeneration in Parkinsonrsquos diseaserdquo Science vol 302 no5646 pp 819ndash822 2003

[4] M Gerlach and P Riederer ldquoAnimal models of Parkinsonrsquosdisease an empirical comparison with the phenomenology ofthe disease in manrdquo Journal of Neural Transmission vol 103 no8-9 pp 987ndash1041 1996

[5] J Lotharius L L Dugan and K L OrsquoMalley ldquoDistinct mech-anisms underlie neurotoxin-mediated cell death in cultureddopaminergic neuronsrdquo Journal of Neuroscience vol 19 no 4pp 1284ndash1293 1999

[6] M K Borland P A Trimmer J D Rubinstein et al ldquoChroniclow-dose rotenone reproduces lewy neurites found in earlystages of Parkinsonrsquos disease reduces mitochondrial movementand slowly kills differentiated SH-SY5Y neural cellsrdquoMolecularNeurodegeneration vol 3 no 1 article 21 2008

[7] NW Kowall P Hantraye E Brouillet M F Beal A C McKeeand R J Ferrante ldquoMPTP induces alpha-synuclein aggregationin the substantia nigra of baboonsrdquo NeuroReport vol 11 no 1pp 211ndash213 2000

[8] N Xiong J Huang Z Zhang et al ldquoStereotaxical infusion ofrotenone a reliable rodent model for Parkinsonrsquos diseaserdquo PLoSONE vol 4 no 11 Article ID e7878 2009

[9] D Blum S Torch N Lambeng et al ldquoMolecular pathwaysinvolved in the neurotoxicity of 6-OHDA dopamine andMPTP contribution to the apoptotic theory in Parkinsonrsquosdiseaserdquo Progress in Neurobiology vol 65 no 2 pp 135ndash1722001

[10] A Panov SDikalovN ShalbuyevaG Taylor T Sherer and J TGreenamyre ldquoRotenone model of Parkinson disease multiple


brain mitochondria dysfunctions after short term systemicrotenone intoxicationrdquo Journal of Biological Chemistry vol 280no 51 pp 42026ndash42035 2005

[11] S Jana M Sinha D Chanda et al ldquoMitochondrial dysfunc-tion mediated by quinone oxidation products of dopamineimplications in dopamine cytotoxicity and pathogenesis ofParkinsonrsquos diseaserdquo Biochimica et Biophysica Acta MolecularBasis of Disease vol 1812 no 6 pp 663ndash673 2011

[12] C S Lee J H Han Y Y Jang J H Song and E S HanldquoDifferential effect of catecholamines and MPP+ on membranepermeability in brain mitochondria and cell viability in PC12cellsrdquo Neurochemistry International vol 40 no 4 pp 361ndash3692002

[13] A H Stokes D Y Lewis L H Lash et al ldquoDopamine toxicityin neuroblastoma cells role of glutathione depletion byL-BSOand apoptosisrdquo Brain Research vol 858 no 1 pp 1ndash8 2000

[14] C-T Lai and P H Yu ldquoDopamine- and L-120573-34-dihydroxyphenylalanine hydrochloride (L-DOPA)-inducedcytotoxicity towards catecholaminergic neuroblastoma SH-SY5Y cells effects of oxidative stress and antioxidative factorsrdquoBiochemical Pharmacology vol 53 no 3 pp 363ndash372 1997

[15] G Walkinshaw and C M Waters ldquoInduction of apoptosis incatecholaminergic PC12 cells by L-DOPA implications for thetreatment of Parkinsonrsquos diseaserdquo Journal of Clinical Investiga-tion vol 95 no 6 pp 2458ndash2464 1995

[16] Y C Youn O S Kwon E S Han J H Song Y K Shin andC S Lee ldquoProtective effect of boldine on dopamine-inducedmembrane permeability transition in brain mitochondria andviability loss in PC12 cellsrdquo Biochemical Pharmacology vol 63no 3 pp 495ndash505 2002

[17] P Weingarten J Bermak and Q-Y Zhou ldquoEvidence for non-oxidative dopamine cytotoxicity potent activation of NF-120581 Band lack of protection by anti-oxidantsrdquo Journal of Neurochem-istry vol 76 no 6 pp 1794ndash1804 2001

[18] R Pedrosa andP Soares-da-Silva ldquoOxidative and non-oxidativemechanisms of neuronal cell death and apoptosis by L-34-dihydroxyphenylalanine (L-DOPA) and dopaminerdquo BritishJournal of Pharmacology vol 137 no 8 pp 1305ndash1313 2002

[19] K S P McNaught R Belizaire P Jenner C W Olanow andO Isacson ldquoSelective loss of 20S proteasome 120572-subunits inthe substantia nigra pars compacta in Parkinsonrsquos diseaserdquoNeuroscience Letters vol 326 no 3 pp 155ndash158 2002

[20] K S P McNaught R Belizaire O Isacson P Jenner and C WOlanow ldquoAltered proteasomal function in sporadic ParkinsonrsquosdiseaserdquoExperimentalNeurology vol 179 no 1 pp 38ndash46 2003

[21] W Zhou M S Huribert J Schaack K N Prasad andC R Freed ldquoOverexpression of human 120572-synuclein causesdopamine neuron death in rat primary culture and immortal-ized mesencephalon-derived cellsrdquo Brain Research vol 866 no1-2 pp 33ndash43 2000

[22] S J Tabrizi M Orth J Max Wilkinson et al ldquoExpression ofmutant120572-synuclein causes increased susceptibility to dopaminetoxicityrdquo Human Molecular Genetics vol 9 no 18 pp 2683ndash2689 2000

[23] M Bisaglia E Greggio D Maric D W Miller M R Cooksonand L Bubacco ldquoAlpha-synuclein overexpression increasesdopamine toxicity in BE2-M17 cellsrdquo BMC neuroscience vol 11article 41 2010

[24] T Hasegawa M Matsuzaki-Kobayashi A Takeda et al ldquo120572-Synuclein facilitates the toxicity of oxidized catechol metabo-lites implications for selective neurodegeneration in Parkin-sonrsquos diseaserdquo FEBS Letters vol 580 no 8 pp 2147ndash2152 2006

[25] K BanerjeeM Sinha C L L Phamet al ldquo120572-Synuclein inducedmembrane depolarization and loss of phosphorylation capacityof isolated rat brain mitochondria Implications in Parkinsonrsquosdiseaserdquo FEBS Letters vol 584 no 8 pp 1571ndash1576 2010

[26] J Y Sung J Kim S R Paik J H Park Y S Ahn and K CChung ldquoInduction of neuronal cell death by Rab5A-dependentendocytosis of120572-synucleinrdquo Journal of Biological Chemistry vol276 no 29 pp 27441ndash27448 2001

[27] O Yermolaieva R Xu C Schinstock et al ldquoMethioninesulfoxide reductase A protects neuronal cells against briefhypoxiareoxygenationrdquo Proceedings of the National Academy ofSciences of the United States of America vol 101 no 5 pp 1159ndash1164 2004

[28] M Kitazawa V Anantharam A Kanthasamy and A G Kan-thasamy ldquoDieldrin promotes proteolytic cleavage of poly(ADP-ribose) polymerase and apoptosis in dopaminergic cells pro-tective effect of mitochondrial anti-apoptotic protein Bcl-2rdquoNeuroToxicology vol 25 no 4 pp 589ndash598 2004

[29] J G Mohanty J S Jaffe E S Schulman and D G Raible ldquoAhighly sensitive fluorescent micro-assay of H

2O2release from

activated human leukocytes using a dihydroxyphenoxazinederivativerdquo Journal of Immunological Methods vol 202 no 2pp 133ndash141 1997

[30] F H Khan M Saha and S Chakrabarti ldquoDopamine inducedprotein damage in mitochondrial-synaptosomal fraction of ratbrainrdquo Brain Research vol 895 no 1-2 pp 245ndash249 2001

[31] H J Choi S W Kim S Y Lee and O Hwang ldquoDopamine-dependent cytotoxicity of tetrahydrobiopterin a possiblemech-anism for selective neurodegeneration in Parkinsonrsquos diseaserdquoJournal of Neurochemistry vol 86 no 1 pp 143ndash152 2003

[32] T B Sherer R Betarbet A K Stout et al ldquoAn in vitro modelof Parkinsonrsquos disease linking mitochondrial impairment toaltered 120572-synuclein metabolism and oxidative damagerdquo Journalof Neuroscience vol 22 no 16 pp 7006ndash7015 2002

[33] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of biological chemistry vol 193 no 1 pp 265ndash275 1951

[34] D G Altman and J M Bland ldquoStandard deviations andstandard errorsrdquo British Medical Journal vol 331 no 7521article 903 2005

[35] B Pardo M A Mena S Fahn and J G de Yebenes ldquoAscorbicacid protects against levodopa-induced neurotoxicity on acatecholamine-rich human neuroblastoma cell linerdquoMovementDisorders vol 8 no 3 pp 278ndash284 1993

[36] B Pardo M A Mena M J Casarejos C L Paino and J GDe Yebenes ldquoToxic effects of L-DOPA on mesencephalic cellcultures protection with antioxidantsrdquo Brain Research vol 682no 1-2 pp 133ndash143 1995

[37] D Offen I Ziv H Sternin EMelamed andAHochman ldquoPre-vention of dopamine-induced cell death by thiol antioxidantspossible implications for treatment of Parkinsonrsquos diseaserdquoExperimental Neurology vol 141 no 1 pp 32ndash39 1996

[38] D M Kuhn and R Arthur Jr ldquoDopamine inactivates tryp-tophan hydroxylase and forms a redox-cycling quinoproteinpossible endogenous toxin to serotonin neuronsrdquo Journal ofNeuroscience vol 18 no 18 pp 7111ndash7117 1998

[39] D M Kuhn R E Arthur Jr D MThomas and L A ElferinkldquoTyrosine hydroxylase is inactivated by catechol-quinones andconverted to a redox-cycling quinoprotein possible relevanceto Parkinsonrsquos diseaserdquo Journal of Neurochemistry vol 73 no 3pp 1309ndash1317 1999


[40] F H Khan T Sen and S Chakrabarti ldquoDopamine oxi-dation products inhibit Na+ K+-ATPase activity in crudesynaptosomal-mitochondrial fraction from rat brainrdquo FreeRadical Research vol 37 no 6 pp 597ndash601 2003

[41] C Gomez-Santos M Barrachina P Gimenez-Xavier EDalfo I Ferrer and S Ambrosio ldquoInduction of CEBP120573 andGADD153 expression by dopamine in human neuroblastomacells relationship with 120572-synuclein increase and cell damagerdquoBrain Research Bulletin vol 65 no 1 pp 87ndash95 2005

[42] A Jowaed I Schmitt O Kaut and U Wullner ldquoMethyla-tion regulates alpha-synuclein expression and is decreased inParkinsonrsquos disease patientsrsquo brainsrdquo Journal of Neurosciencevol 30 no 18 pp 6355ndash6359 2010

[43] M S Parihar A Parihar M Fujita M Hashimoto andP Ghafourifar ldquoAlpha-synuclein overexpression and aggrega-tion exacerbates impairment of mitochondrial functions byaugmenting oxidative stress in human neuroblastoma cellsrdquoInternational Journal of Biochemistry and Cell Biology vol 41no 10 pp 2015ndash2024 2009

[44] H Hayashita-Kinoh M Yamada T Yokota Y Mizuno andHMochizuki ldquoDown-regulation of120572-synuclein expression canrescue dopaminergic cells fromcell death in the substantia nigraof Parkinsonrsquos disease rat modelrdquo Biochemical and BiophysicalResearch Communications vol 341 no 4 pp 1088ndash1095 2006

[45] M Hashimoto L J Hsu E Rockenstein T Takenouchi MMallory and EMasliah ldquo120572-synuclein protects against oxidativestress via inactivation of the c-Jun N-terminal kinase stress-signaling pathway in neuronal cellsrdquo Journal of BiologicalChemistry vol 277 no 13 pp 11465ndash11472 2002

[46] R E J Musgrove A E King and T C Dickson ldquoNeuropro-tective upregulation of endogenous alpha-synuclein precedesubiquitination in cultured dopaminergic neuronsrdquo Neurotoxi-city Research vol 19 no 4 pp 592ndash602 2011

[47] M R Cookson ldquoalpha-Synuclein and neuronal cell deathrdquoMolecular Neurodegeneration vol 4 article 9 2009

[48] J-H Seo J-C Rah S H Choi et al ldquoAlpha-synuclein regulatesneuronal survival via Bcl-2 family expression and PI3Aktkinase pathwayrdquo The FASEB Journal vol 16 no 13 pp 1826ndash1828 2002

[49] L Shehadeh G Mitsi N Adi N Bishopric and S Papa-petropoulos ldquoExpression of Lewy body protein septin 4 inpostmortem brain of Parkinsonrsquos disease and control subjectsrdquoMovement Disorders vol 24 no 2 pp 204ndash210 2009

[50] J Wills J Jones T Haggerty V Duka J N Joyce andA Sidhu ldquoElevated tauopathy and alpha-synuclein pathologyin postmortem Parkinsonrsquos disease brains with and withoutdementiardquo Experimental Neurology vol 225 no 1 pp 210ndash2182010

[51] MG Spillantini R A Crowther R JakesMHasegawa andMGoedert ldquo120572-Synuclein in filamentous inclusions of Lewy bodiesfrom Parkinsonrsquos disease and dementia with Lewy bodiesrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 95 no 11 pp 6469ndash6473 1998

[52] T L Edwards W K Scott C Almonte et al ldquoGenome-Wideassociation study confirms SNPs in SNCA and the MAPTregion as common risk factors for parkinson diseaserdquo Annalsof Human Genetics vol 74 no 2 pp 97ndash109 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


PD [4ndash8] Although in some models various toxins like6-hydroxydopamine (6-OHDA) rotenone and 1-methyl-4-phenyl-1236-tetrahydropyridine (MPTP) have been used toinduce cell death the cytotoxic action of dopamine (DA)on catecholaminergic cell lines is of special relevance in thecontext of PD pathogenesis because DA is endogenouslyavailable in the body [5 6 9 10] A large body of evidencehas already implicated that DA oxidation products likereactive oxygen species (ROS) and toxic quinones can triggerapoptotic death in cultured PC12 and SH-SY5Y cells inwhich mitochondrial dysfunction plays a critical role [11ndash16] Although most of these studies on DA cytotoxicity havesuggested the oxidative mode of cellular damage a few havealso indicated the existence of a nonoxidative pathway in thisprocess [17 18]

The involvement of proteasomal impairment with abnor-mal accumulation of proteins especially alpha-synucleinin PD pathogenesis has been suggested based on multiplelines of evidence in sporadic and familial PD [19 20] Asubstantial body of evidence also indicates the toxic effectsof alpha-synuclein on different cell lines or on isolated brainmitochondria [21ndash26] Since proteasomal inactivation andalpha-synuclein accumulation occur in the substantia nigralneurons in PD it is likely that these phenomena have someunknown links with dopamine We hypothesize that alpha-synuclein accumulation and proteasomal inactivationmay belinked with either the oxidative or nonoxidative mode of DAtoxicity which could be tested in a cell-based model In thepresent study we have therefore exposed SH-SY5Y cells (aneuroblastoma cell line with the robust expression of alpha-synuclein) to varying concentrations of DA over a periodof 24 h with or without radical and quinone scavengers andmeasured cytotoxicity parameters as well as alpha-synucleincontent and proteasomal activity

2 Materials

The reagents used in the studywere obtained from the follow-ing sources DulbeccorsquosModified EaglersquosMediumNutrient F-12Ham (DMEMF-12Ham 1 1) fetal bovine serum (FBS)551015840661015840-tetrachloro-111015840331015840-tetraethylbenzimidazolocarb-ocyanine iodide (JC-1) ATP bioluminescent assay kit peni-cillin streptomycin and amphotericin B fromSigmaAldrichUSA dopamine (DA) N-acetylcysteine (NAC) and AmplexRed from Acros Organics USA fluorogenic peptides (Suc-Leu-Leu-Val-Tyr-AMC and Z-Leu-Leu-Glu-AMC) and lacta-cystin from Alexis Biochemicals USA nitroblue tetrazolium(NBT) from Promega India polyclonal rabbit anti-alpha-synuclein antibody from Santa Cruz Biotechnology USAhorseradish peroxidase- (HRP-) conjugated secondary goatanti-rabbit IgG antibody from Bangalore Genei India RNAextraction kit from Roche Applied Science Germany andreverse transcription kit forward and reverse primers forboth the target (alpha-synuclein) and the reference (120573-actin) genes from Roche Diagnostics Germany All commonchemicals and reagents used were of the highest analyticalgrade available and were obtained from Sisco ResearchLaboratories India

3 Methods

31 Cell Culture and Treatment Paradigm Undifferentiatedhuman neuroblastoma SH-SY5Y cells obtained from Amer-ican Type Culture Collection (ATCC USA) were grownin DMEMF-12Ham containing 10 (vv) heat-inactivatedFBS 50 unitsmL penicillin 50 120583gmL streptomycin and25 120583gmL amphotericin B in a humidified environmentcontaining 5 CO

2and 95 air at 37∘C in 25 cm2 sterile

tissue culture flasks SH-SY5Y cells (70ndash75 confluent) wereincubated with or without varying concentrations of DA(100ndash400 120583M) in the absence or presence of other additionslike NAC (25mM) or lactacystin (5 120583M) for 24 h Controland treated cells were analyzed for cell death mitochondrialfunctions oxidative stress parameters proteasomal activ-ity and alpha-synuclein content and alpha-synuclein geneexpression

32 Assessment of Cell Death

321 Trypan Blue Assay An aliquot of cell suspension inphosphate-buffered saline (PBS) was mixed with an equalvolume of trypan blue solution (04 in PBS) and the cellswere counted in a Neubauer chamber The cell death wasexpressed as the percentage of trypan blue positive cells in thetotal population of stained and unstained cells counted by anobserver blind to treatment protocol [27]

322 Lactate Dehydrogenase (LDH) Assay Cell death wasalso measured by LDH assay in which the activity of LDHreleased in the medium expressed as the percentage of thetotal LDH (intracellular plus extracellular) was measuredusing NADH-based spectrophotometric assay [28]

33 Measurement of Mitochondrial Membrane Potential inSH-SY5YCells Mitochondrial transmembrane potential wasmeasured in intact SH-SY5Y cells by using a cationic carbo-cyanine dye JC-1 which remains distributed in the cytosolas monomers while after entering the mitochondria drivenby the electrochemical gradient undergoes concentration-dependent aggregation to form J-aggregates The monomersemit a green fluorescence (120582ex 490 nm 120582em 530 nm) and J-aggregates a red fluorescence (120582ex 490 nm 120582em 590 nm) andthe ratio of fluorescence intensities at 590 nm to 530 nm indi-cates mitochondrial transmembrane potential [11] Brieflycontrol and treated cells were washed twice in PBS andthen incubated in serum-free DMEM for 30min in thepresence of JC-1 (10 120583M) at 37∘C in the dark The cellswere pelleted down and then washed thrice with serum-free DMEM suspended with suitable dilutions in the samemedium and the fluorescence emission intensities (590 nmand 530 nm) were measured with excitation at 490 nm ina spectrofluorometer (FP-6300 JASCO International CoJapan)

34 Measurement of ATP Content in SH-SY5Y Cells TheATP content was measured in control and treated SH-SY5Y cells by using a commercial kit based on luciferin-luciferase reaction as described previously [11] Briefly the

















0

10

20

30

40

50

60

70

80

90

100C

ell d

eath

(per

cent

age o

f try

pan

blue

pos

itive

cells

)lowast

lowast

lowast

lowast

lowast


DA(400120583M)

DA(100120583M)

DA(400120583M)+ NAC

(a)

0

10

20

30

40

50

60

70

80

90

100

LDH

rele

ased

()

lowast

lowast

lowast

lowast

lowast


DA(400120583M)

DA(100120583M)

DA(400120583M)+ NAC

(b)


0

2

4

6

8

10

12

14

16

18

JC-1

fluo

resc

ence

(590

nm5

27 nm

ratio

)

lowast

lowastlowast


DA(400120583M)+ NAC

(a)

0

20

40

60

80

100

120

Intr

acel

lula

r ATP

cont

ent (

perc

enta

ge o

f con

trol)

lowast

lowast

lowast


DA(400120583M)+ NAC

(b)



0

50

100

150

200

250

300

350

nmol

of h

ydro

gen

pero

xide

per

mg

of p

rote

in

lowastlowast


DA(400120583M)+ NAC(25 mM)

(a)

0

01

02

03

04

05

06

Prot

ein

boun

d qu

inon

es (O

D 5

30m

g pr

otei

n)

lowast lowast


DA(400120583M)+ NAC(25 mM)

(b)






4 Results







0

20

40

60

80

100

120Pr

otea

som

al ac

tivity

(per

cent

age o

f con

trol)


DA(400120583M)+ NAC

lowast

lowast

lowast






0

5

10

15

20

25

30

35

Control Lactacystin

Cel

l dea

th (p

erce

ntag

e of t

rypa

n bl

ue p

ositi

ve ce

lls)

lowast

(a)

0

5

10

15

20

25

30

35

40

LDH

rele

ased

()

Control Lactacystin

lowast

(b)




5 Discussion



0

2

4

6

8

10

12

14

16

Control Lactacystin

lowast

JC-1

fluo

resc

ence

(590

nm5

27 nm

ratio

)

(a)

0

20

40

60

80

100

120

Control Lactacystin

lowast

Intr

acel

lula

r ATP

cont

ent (

perc

enta

ge o

f con

trol)

(b)










120572-Synuclein

120574-Actin

(a)

0

5

10

15


Fold

s of120572

-syn

ucle

in p

rote

in ex

pres

sion

(au

) lowast

lowast

lowastlowast

(b)


0

05

1

15

2

25

3

35

4

45

Fold

incr

ease

of120572

-syn

ucle

in g

ene e

xpre

ssio

n

DA(400120583M)

DA(400120583M)+ NAC

lowast





Cell death





(a)


cells






uarr

darr

degradationuarr

accumulation

gene expression


120572-Synuclein

(b)



Cell death


Lactacystin


(c)


6 Conclusions





Abbreviations











Acknowledgments


References
































2O2release from































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
































0

10

20

30

40

50

60

70

80

90

100C

ell d

eath

(per

cent

age o

f try

pan

blue

pos

itive

cells

)lowast

lowast

lowast

lowast

lowast


DA(400120583M)

DA(100120583M)

DA(400120583M)+ NAC

(a)

0

10

20

30

40

50

60

70

80

90

100

LDH

rele

ased

()

lowast

lowast

lowast

lowast

lowast


DA(400120583M)

DA(100120583M)

DA(400120583M)+ NAC

(b)


0

2

4

6

8

10

12

14

16

18

JC-1

fluo

resc

ence

(590

nm5

27 nm

ratio

)

lowast

lowastlowast


DA(400120583M)+ NAC

(a)

0

20

40

60

80

100

120

Intr

acel

lula

r ATP

cont

ent (

perc

enta

ge o

f con

trol)

lowast

lowast

lowast


DA(400120583M)+ NAC

(b)



0

50

100

150

200

250

300

350

nmol

of h

ydro

gen

pero

xide

per

mg

of p

rote

in

lowastlowast


DA(400120583M)+ NAC(25 mM)

(a)

0

01

02

03

04

05

06

Prot

ein

boun

d qu

inon

es (O

D 5

30m

g pr

otei

n)

lowast lowast


DA(400120583M)+ NAC(25 mM)

(b)






4 Results







0

20

40

60

80

100

120Pr

otea

som

al ac

tivity

(per

cent

age o

f con

trol)


DA(400120583M)+ NAC

lowast

lowast

lowast






0

5

10

15

20

25

30

35

Control Lactacystin

Cel

l dea

th (p

erce

ntag

e of t

rypa

n bl

ue p

ositi

ve ce

lls)

lowast

(a)

0

5

10

15

20

25

30

35

40

LDH

rele

ased

()

Control Lactacystin

lowast

(b)




5 Discussion



0

2

4

6

8

10

12

14

16

Control Lactacystin

lowast

JC-1

fluo

resc

ence

(590

nm5

27 nm

ratio

)

(a)

0

20

40

60

80

100

120

Control Lactacystin

lowast

Intr

acel

lula

r ATP

cont

ent (

perc

enta

ge o

f con

trol)

(b)










120572-Synuclein

120574-Actin

(a)

0

5

10

15


Fold

s of120572

-syn

ucle

in p

rote

in ex

pres

sion

(au

) lowast

lowast

lowastlowast

(b)


0

05

1

15

2

25

3

35

4

45

Fold

incr

ease

of120572

-syn

ucle

in g

ene e

xpre

ssio

n

DA(400120583M)

DA(400120583M)+ NAC

lowast





Cell death





(a)


cells






uarr

darr

degradationuarr

accumulation

gene expression


120572-Synuclein

(b)



Cell death


Lactacystin


(c)


6 Conclusions





Abbreviations











Acknowledgments


References
































2O2release from































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

50

100

150

200

250

300

350

nmol

of h

ydro

gen

pero

xide

per

mg

of p

rote

in

lowastlowast


DA(400120583M)+ NAC(25 mM)

(a)

0

01

02

03

04

05

06

Prot

ein

boun

d qu

inon

es (O

D 5

30m

g pr

otei

n)

lowast lowast


DA(400120583M)+ NAC(25 mM)

(b)






4 Results







0

20

40

60

80

100

120Pr

otea

som

al ac

tivity

(per

cent

age o

f con

trol)


DA(400120583M)+ NAC

lowast

lowast

lowast






0

5

10

15

20

25

30

35

Control Lactacystin

Cel

l dea

th (p

erce

ntag

e of t

rypa

n bl

ue p

ositi

ve ce

lls)

lowast

(a)

0

5

10

15

20

25

30

35

40

LDH

rele

ased

()

Control Lactacystin

lowast

(b)




5 Discussion



0

2

4

6

8

10

12

14

16

Control Lactacystin

lowast

JC-1

fluo

resc

ence

(590

nm5

27 nm

ratio

)

(a)

0

20

40

60

80

100

120

Control Lactacystin

lowast

Intr

acel

lula

r ATP

cont

ent (

perc

enta

ge o

f con

trol)

(b)










120572-Synuclein

120574-Actin

(a)

0

5

10

15


Fold

s of120572

-syn

ucle

in p

rote

in ex

pres

sion

(au

) lowast

lowast

lowastlowast

(b)


0

05

1

15

2

25

3

35

4

45

Fold

incr

ease

of120572

-syn

ucle

in g

ene e

xpre

ssio

n

DA(400120583M)

DA(400120583M)+ NAC

lowast





Cell death





(a)


cells






uarr

darr

degradationuarr

accumulation

gene expression


120572-Synuclein

(b)



Cell death


Lactacystin


(c)


6 Conclusions





Abbreviations











Acknowledgments


References
































2O2release from































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

20

40

60

80

100

120Pr

otea

som

al ac

tivity

(per

cent

age o

f con

trol)


DA(400120583M)+ NAC

lowast

lowast

lowast






0

5

10

15

20

25

30

35

Control Lactacystin

Cel

l dea

th (p

erce

ntag

e of t

rypa

n bl

ue p

ositi

ve ce

lls)

lowast

(a)

0

5

10

15

20

25

30

35

40

LDH

rele

ased

()

Control Lactacystin

lowast

(b)




5 Discussion



0

2

4

6

8

10

12

14

16

Control Lactacystin

lowast

JC-1

fluo

resc

ence

(590

nm5

27 nm

ratio

)

(a)

0

20

40

60

80

100

120

Control Lactacystin

lowast

Intr

acel

lula

r ATP

cont

ent (

perc

enta

ge o

f con

trol)

(b)










120572-Synuclein

120574-Actin

(a)

0

5

10

15


Fold

s of120572

-syn

ucle

in p

rote

in ex

pres

sion

(au

) lowast

lowast

lowastlowast

(b)


0

05

1

15

2

25

3

35

4

45

Fold

incr

ease

of120572

-syn

ucle

in g

ene e

xpre

ssio

n

DA(400120583M)

DA(400120583M)+ NAC

lowast





Cell death





(a)


cells






uarr

darr

degradationuarr

accumulation

gene expression


120572-Synuclein

(b)



Cell death


Lactacystin


(c)


6 Conclusions





Abbreviations











Acknowledgments


References
































2O2release from































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

2

4

6

8

10

12

14

16

Control Lactacystin

lowast

JC-1

fluo

resc

ence

(590

nm5

27 nm

ratio

)

(a)

0

20

40

60

80

100

120

Control Lactacystin

lowast

Intr

acel

lula

r ATP

cont

ent (

perc

enta

ge o

f con

trol)

(b)










120572-Synuclein

120574-Actin

(a)

0

5

10

15


Fold

s of120572

-syn

ucle

in p

rote

in ex

pres

sion

(au

) lowast

lowast

lowastlowast

(b)


0

05

1

15

2

25

3

35

4

45

Fold

incr

ease

of120572

-syn

ucle

in g

ene e

xpre

ssio

n

DA(400120583M)

DA(400120583M)+ NAC

lowast





Cell death





(a)


cells






uarr

darr

degradationuarr

accumulation

gene expression


120572-Synuclein

(b)



Cell death


Lactacystin


(c)


6 Conclusions





Abbreviations











Acknowledgments


References
































2O2release from































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















120572-Synuclein

120574-Actin

(a)

0

5

10

15


Fold

s of120572

-syn

ucle

in p

rote

in ex

pres

sion

(au

) lowast

lowast

lowastlowast

(b)


0

05

1

15

2

25

3

35

4

45

Fold

incr

ease

of120572

-syn

ucle

in g

ene e

xpre

ssio

n

DA(400120583M)

DA(400120583M)+ NAC

lowast





Cell death





(a)


cells






uarr

darr

degradationuarr

accumulation

gene expression


120572-Synuclein

(b)



Cell death


Lactacystin


(c)


6 Conclusions





Abbreviations











Acknowledgments


References
































2O2release from































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Cell death





(a)


cells






uarr

darr

degradationuarr

accumulation

gene expression


120572-Synuclein

(b)



Cell death


Lactacystin


(c)


6 Conclusions





Abbreviations











Acknowledgments


References
































2O2release from































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Abbreviations











Acknowledgments


References
































2O2release from































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





































2O2release from































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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