Terminally Differentiated SH-SY5Y Cells Provide a Model [887035]

8/18/2019 Terminally Differentiated SH-SY5Y Cells Provide a Model [887035]

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We characterized undifferentiated (UN) and threedifferentiation conditions of the SH-SY5Y neurob-

lastoma cell line for phenotypic markers of dopaminergic cells, sensitivity to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridinium ion(MPP ), the requirement to utilize the dopamine(DA) transporter (DAT) for MPP + toxicity, and theneuroprotective effects of pramipexole. Cells weredifferentiated with retinoic acid (RA), 12- O-tetra e-canoyl-phorbol-13-acetate (TPA), and RA followedby TPA (RA/TPA). RA/TPA treated cells exhibitedthe highest levels of tyrosine hydroxylase and DATbut lower levels of vesicular monoamine trans-porter. The kinetics of [ 3H]DA uptake and [ H]MPP +

uptake to DAT in RA/TPA differentiated cells weresimilar to that of rat and mouse caudate-putamensynaptosomes. RA/TPA differentiated cells evi-denced high sensitivity to the neurotoxic effects of MPP + (0.03 to 3.0 mM , and the neurotoxic effects of

+ were blocked with the DAT inhibitor 1-(2-[ is (4-fluorophenyl)methoxy]ethyl)-4-(3-phenyl-propyl)piperazine (GBR 12909). DA-induced celldeath was not more sensitive in RA vs RA/TPA dif-ferentiated cells and was not inhibited by trans-porter inhibitors. RA/TPA differentiated cells

exhibited 3- fold and 6-fold higher levels, respec-tively, of DA D 2 and D 3 receptors than UN or RA dif-ferentiated cells. Pretreatment with pramipexolewas protective against MPP + in the RA/TPA differ-entiated cells but not in undifferentiated or RA dif-ferentiated cells. The neuroprotective effect of pramipexole was concentration-dependent anddopamine D 2/D 3 receptor epen ent. n contrast,protection by pramipexole against DA was not DAreceptor dependent. Further characterization of the

neuroprotective effects of DA agonists in this modelsystem can provide unique information about DA

receptor dependent and independent mechanismsof neuroprotection.

Keywor s: Par nson's sease; D Receptor; D Receptor;MPP +; Dopam ne transporter; Pram pexo e

INTRODUCTION

Parkinson's disease (PD) is a neurodegenerative disor-er with an insidious onset and a prolonged course over

many years. The primary cause of the symptoms of thisness s t e eat of opam ne- DA- pro uc ng neu-

rons of the substantia nigra (SN) and the resultantep et on of DA n t e str atum Horny ew cz, 1998 .

W e -3,4- y roxyp eny a an ne - opa pro-vides effective symptomatic treatment for PD, it doesnot a ter t e progress on of t e sease. DAagon sts arealso used for treatment of PD, some of which have

been shown to be neuroprotective in Parkinson's mod-els (Anderson et al. , 2001) and, perhaps, in PD itself Mare et al. , 2002 . Ant par nson an agents t at are

direct DA agonists such as apomorphine (Grünblatt et

al. , 1999 , romocr pt ne Mura r s nan anMo ana umar, 1998 , an pram pexo e K tamura et al. , 1997 , have been shown to be neuroprotectiveaga nst 1-met y -4-p eny -1,2,3,6-tetra y ropyr ne-MPTP- n uce amage to t e DA system n m ce.

MPTP administration to mice is considered a goodmodel of studying neuroprotection because MPTP is

nown to pro uce par nson sm n umans an n su -human species through selective loss of DA-ergic neu-rons of t e SN Burns et al. , 1983; Langston et al. ,

F.P. ra am u s ng Co.

Neurotoxicity Research, 2004, VOL. 5(8). pp. 579-598

Terminally Differentiated SH-SY5Y Cells Provide a ModelSystem for Studying Neuroprotective Effects of DopamineAgonistsSTEVEN P. PRESGRAVES a, TARIQ AHMED , SABINE BORWEGE and JEFFREY N. JOYCE ,

o ecu ar an e u ar io ogy ra uate roup, rizona tate niversity, empe, ; an homas H. Christopher Center for Parkinson's Disease Research Center, Sun Health Research Institute, 10515 West Santa Fe Dr., Sun City; AZ 85351. [email protected]

(Receive 28 January 2004; Revise 27 Fe ruary 2004; In fina form 27 Fe ruary 2004)

*Corresponding author. Tel: +1 623 876-5439; Fax: 1 623 876-5695; E-mail: [email protected] 1029 8428 print/ ISSN 1476-3524 online. © 2004 FP Graham Publishing Co., www.fpgrahamco.com


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S.P. PRESGRAVES t al.580

.MPTP that produce nigral cell loss in primatesMcNaug t et al. , 1996 . MPTP pro uces apoptot c

c anges assoc ate w t PD Co en an Werner, 1994;Cassarino and Bennett Jr., 1999; Hirsch et al. , 2000 ;an MPTP pro uces ongo ng ce eat n umans for

eca es after t e n t a nsu t Langston et al. , 1999 .Hence, drugs that reduce the neurotoxicity of com-

poun s suc as MPTP, or ts act ve meta o te MPP +

may prove to be neuroprotective in PD.However, the efficacy of current DA agonists in neu-

roprotection is less than what is desirable and an under-stan ng of t e mec an sms of protect on m g t pro-vide better targets. Because the DA D receptor prefer-r ng agon sts pram pexo e an rop n ro e appear to ethe most potent of the agonists in vivo (Kitamura et al. ,1997; I a et al. , 1999; Zou et al. , 2000 t as suggest-e t at t e D receptor p ays an mportant ro e. T s as

been further substantiated by the findings that the neu-

roprotect ve effects of pram pexo e aga nst MPTP sreduced in D 3 receptor knockout mice Ramirez et al. ,2003 . Ce cu tures ave t e a vantage of ent fy ngeven more specifically the receptor and nonreceptor mediated effects of drugs and it has been establised that

pram pexo e can prevent neurotox c ty pro uce y L-opa in mesencephalic cultures (Carvey et al. , 1997;

Ling et al. , 1999) and by MPP + n the neuro lastomaSH-SY5Y ce ne K tamura et al. , 1998 . Carvey anassociates have provided valuable evidence the Dreceptor mediates a ma or component of the effects of

pramipexole (Carvey et al. , 2001). In contrast, another group has shown that blockade of DA receptors witheither D 2-selective or non-selective compounds did not nhibit the neuroprotective effects of talipexole or

pramipexole against MPP +-induced apoptosis of SH-SY5Y ce s K tamura et al. , 1998 . T s apparent con-flict might reflect differences in the cell culture systemuse , or t e tox n ut ze .

W e mesencep a c cu tures ave t e a vantage of being a source of DA neurons, L-dopa induced neuro-tox c ty may not ut ze t e same ce eat pat ways asMPP + Murer et al. , 1998; C o et al. , 1999; Kos muraet al. , 2000; Blum et al. , 2001 . MPP +, which has beenuse n t e SH-SY5Y ce s, oes m m c many aspectsof t e DA neuron eat o serve n PD. T e umanneuroblastoma cell line SH-SY5Y, which was sub-c one from t e SK-N-SH ce ne; ex ts neurona

properties and a catecholaminergic phenotype includ-ng the ability to transport DA and NE, express

enzymes for t e synt es s an meta o sm of DA anacety c o ne, an express receptors for DAan acety -choline Biedler et al. , 1978; Ross et al. , 1983 ; Willets

., . -igh concentrations of MPP or 3 days will produce

ce eat w t c ear morp o og ca ev ence of apop-tos s Fang et al. , 1995; S ee an et al. , 1997; Song et al. , 1997; Kitamura et al. , 1998 . However, the con-centrat ons of MPP + t at are requ re to pro uce neu-rotox c ty n t e un fferent ate state of t s ce neare higher than those needed for mesencephalic cul-tures Storc et al. , 2000a . W e t as een assumethat MPP +-induced cell death required the intracellular accumulation through DAT in the SH-SY5Y cell line(Spina et al. , 1992; Song et al. , 1997; Park et al. , 1998;K tamura et al. , 1998 , as t oes in vivo , t e re at ve y

igh resistance of these cells to this compound wouldsuggest ot erw se. T e requ rement to use re at ve y

g concentrat ons of MPP + to pro uce neurotox c tymay be due to the low expression of the DAT and higexpress on of ves cu ar monoam ne transporter (VMAT) in this cell line in the undifferentiated state.

To test if the insensitivity of the neuroblastoma SH-SY5Y cell line to MPP + and to the DA receptor-medi-ate effects of pram pexo e ref ecte propert es of t eundifferentiated SH-SY5Y cells, we examined this cell

ne after treatment w t factors t at fferent ate t esece s to a p enotyp ca y mature opam nerg c ce type(Pennypacker et al. , 1989 . We compared the neuro-tox c effects of MPP + w t t at of DA w c s t oug tto produce nonapoptotoic death (Gómez-Santos et al. ,2003 . We have identified that SH-SY5Y cells differ-entiated with retinoic acid (RA) and the phorbol ester 12- O-tetradecanoyl-phorbol-13-acetate (TPA) exhibitthe most pronounced dopaminergic phenotype, charac-ter st cs of MPP - n uce tox c ty s m ar to DA neu-rons in vivo , and D -like receptor dependent neuropro-tect on aga nst MPP +

MATERIALS AND METHODS

rugsS - pram pexo e was prov e y P armac aCorporat on Ka amazoo, MI, USA . MPP + o e was

obtained from Research Biochemicals Natick, MA,USA . A ot er c em ca s were purc ase from S gmaChemical Co. (St Louis, MO, USA) unless otherwisestated.

Cell CultureHuman SH-SY5Y neuroblastoma cells (ATCC,Manassas, VA, USA were grown to conf uence n SH-SY5Y me a [Du ecco's Mo f e Eag e's me aDMEM supplemented with 10% fetal calf serum, 100


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SH-SY5Y CELLS AS A MODEL TO STUDY NEUROPROTECTION 581

, , .amp oter c n B G co Gran Is an , NY, USA , an0.01 µM non-essent a am no ac s G co ] an t ensub-cultured for differentiation in 48 well culture platesCorn ng Costar, Corn ng, NY, USA . Four st nct f-

ferent at on protoco s were ut ze : Un fferent atecells (UN) were grown in SH-SY5Y media for 3 days;t en me a was remove an rep ace w t fres SH-SY5Y me a for anot er 3 ays growt . Ret no c ac(RA) differentiated cells were grown in SH-SY5Yme a conta n ng 10 µM RA for 3 ays; t en t e me awas removed and replaced with fresh RA media for another 3 days of differentiation. Phorbol ester differ-ent ate ce s were grown n 80 nM 12- O-tetra e-canoy -p or o -13-acetate TPA for 3 ays; t en t emedia was removed and replaced with fresh TPA mediafor anot er 3 ays of fferent at on. RA TPA fferen-t ate ce s were grown n RA me a for 3 ays; t enthe media was removed and replaced with fresh TPA

me a for anot er 3 ays of fferent at on.

Cytotoxic Effects of MPP + and DA on SH-SY5YCellsSH-SY5Y cells were subcultured into 48 well plates25,000 cells/well . The cells were then differentiate

as a ove for RA fferent ate an RA TPA fferent -ated cells. Each of these populations was then treatewith ifferent concentrations of MPP + 0.0, 0.03, 0.3mM, 1.0, an 3.0 mM or DA 0, 1, 5, 10, 50, 100, 150,250 and 500 µM) in SH-SY5Y media. To test for cellviability and other measures, sister cultures were initi-ated at the same time and condition and stopped at time

point for assessments. Cell viability was assayed usingthe MTT salt assay, LDH assay and cell counts at 24,48, 72 an 96 post MPP

Transporter AntagonistsD fferent ate RA an RA TPA SH-SY5Y ce s werencubated with 1- 2-[ bis (4-fluorophenyl)methoxy]

ethyl)-4-(3-phenylpropyl)piperazine (GBR 12909) inSH-SY5Y me a for 1 pr or to MPP + a m n strat onand then treated with different concentrations of MPP +

(0 - 3.0 mM) in SH-SY5Y media. Cell viability wasassaye us ng t e MTT an LDH assays. RA TPA f-ferentiated SH-SY5Y cells were incubated with 10 µMof the DAT inhibitor GBR 12909, the NE transportn tor, n soxet ne, an t e seroton n 5-HT trans-

port inhibitor 6-nitroquipazine or combinations of thetransport inhibitors for 1 h prior to addition of 200 µMDA, an ce v a ty was measure at 24 nterva s.Cell viability was assayed with cell count, GSH levelsand hydrogen peroxide (H O 2 levels.

D fferent ate RA an RA TPA SH-SY5Y ce s weree t er pretreate w t me a conta n ng pram pexo e(concentration range of 0-1.0 mM) for 3 days thentreate w t MPP + 100 µM conta n ng me a; or con-current y treate w t me a conta n ng pram pexo e(concentration range of 0-1.0 mM) and MPP or DA.SH-SY5Y ce s were su cu ture an fferent ate as

escr e a ove. RA TPA fferent ate ce s wereeither pretreated with media containing 0 µM

pram pexo e, 100 µM pram pexo e, 100 µM pram pex-ole plus 10 µM spiperone, 100 µM pramipexole plus 1µM U99194A [5,6-dimethoxy-2-(di-u-propylaminon an], or 100 µM pram pexo e p us 10 µM SCH

23390 [ R +)-3-methyl-7-chloro-8-hydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine] for 3 days, thentreate w t MPP + 300 µM or DA 150 µM conta n-ng media. Subsequently cell viability was assaye

using the MTT and LDH assays and cell counts at 24

nterva s.

MTT Assay for Cell ViabilityThe cytotoxic affect of the MPP + was assayed over afour-day period at 24 h intervals using the MTT (3-[4,5-Dimethylthiazol]-2,5-diphenyltetrazolium) saltassay as escr e y S gma. MTT stoc so ut on 5mg/ml) was added to each well at one tenth the totamedia volume and left to incubate at 37°C for 4 h. Att e en of t s ncu at on, t e me a was remove anthe converted dye solubilized with acidic isopropanol0.1 N HCl in absolute isopropanol for 4 h.

Absorbance of the dye was measured at 570 nm with a background subtraction at 670 nm. The data were thentotaled, averaged and represented as a percent cell

eat as compare to t e 0.0 mM MPP treatment oeach differentiation condition over the four-day MPP +

reatment.

Lactate Dehydrogenase Assay for Cell ViabilityThe cytotoxic effect of the MPP + was assayed over afour- ay per o at 24 nterva s us ng Lactate e y-

rogenase method (LDH) of activity as a measure o

cell membrane integrity. The Cytotox 96 (Promega)assessment of ce v a ty s a quant tat ve measure olactate dehydrogenase, a stable enzyme, released intothe media upon cell death and is measured colorimetri-ca y ase on t e convers on of actate to pyruvate nthe presence of NAD coupled to the conversion of atetrazolium salt into a red formazan product. Aliquotsof 50 µ of me a from eac we were transferre to a96 well plate. The remaining media was removed, lysis

buffer was added and the plates were incubated for 1


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° . ,each transfer plate. The plates were maintained in the

ar an ncu ate at room temperature for 30 m n.T e react on was stoppe an t e a sor ance of t econverted dye measured at 490 nm. The data weretota e , average , an represente as percent ce

eat .

Cell Count Assessment of Cell ViabilitySH-SY5Y cells were cultured in 48 well culture plates.After the individual treatments, cells were detachedand stained with trypan blue (final concentration,0.023% w v for 3 m n. T e v a e, non-co ore , an

ead, blue-colored, cells were counted from an aliquot1 m of ce suspens on us ng a emocytometer un er

400x magn f cat on. T e ata were ana yze as t eratio of alive to dead cells % cell survival .

Protocol for Extracellular and Intracellular

Glutathione LevelsThe principle of the procedure is based on the oxida-t on of re uce g utat one GSH y 5,5'- t o s 2-nitrobenzoic acid) [DTNB], to measure the total glu-tat one content of a o og ca samp e. So ut ons of DA n t e ce cu ture me a were prepare w t 1 mMGSH added. At different time points, aliquots wereta en to measure t e GSH rema n ng n t e me aextrace u ar GSH . DTNB was fres y prepare pr or

to use. To aliquot of media (50 µl), 50 µl of 10 mMDTNB was added to a 96-well assay plate (6 sam-

p es treatment , ncu ate for 10-15 m n, anabsorbency was read at 405 nm against a DTNB blank w t V ctor Wa ace p ate rea er. For ntrace u ar measures the cells were centrifuged for 10 min to pel-et cells, supernatant was removed and discarded, pel-ets were resuspen e w t 250 µ Tr s-EDTA an

transferre to 1.5 m m crocentr fuge tu es. Ce s wereysed by freezing, then 50 µl samples were transferre

to 96-we p ates 5 samp es treatment an 50 µ of 10mM DTNB was a e , ncu ate for 10-15 m n, anabsorbency was read at 405 nm against a DTNB blank w t V ctor Wa ace p ate rea er. A GSH stan ar con-

centration curve (0.5, 1, 2.5, 5, 10, 15 and 20 µM) wasused as a standard. The data were then totaled, aver-aged and represented as a percent change from the 0.0mM DA treatment.

PeroXOquant Quantitative Peroxide AssayT s assay prov es a quant tat ve measure of H 2O2eve s ase on ox at on of ferrous to ferr c on n t e

presence of xylenol orange. Solutions of DA in the cellculture media were prepared with 1 mM GSH added.

, -ure the H 2O2 evels in the media extracellular H 2O2 .The 20 µl aliquots of experimental media from treatedcells were transferred to a 96-well assay plate (6 sam-

ples/treatment), 200 µl of working solution was addePeroXOquant Quant tat ve Perox e Assay, P erce

Chemical, Rockford, IL, USA , incubated for 20-25m n at room temperature, an a sor ency rea at560 nm w t V ctor Wa ace p ate rea er w t a stan-

ard curve generated from known concentrations oH O T e ata were t en tota e , average an repre-sented as a percent change from the 0.0 mM DA treat-ment.

Intracellular Reactive Oxygen Species LevelsUsing CM-H 2DCFDACM-H 2DCFDA is a cell-permeant indicator for reac-t ve oxygen spec es a T o -React ve tracer t at snonfluorescent until removal of the acetate groups byintracellular esterases, and oxidation occurs within thece . Cytop asm c enzymes y ro yze t e acetategroups from this membrane-permeant probe and thechloromethyl moieties become con ugated to intracel-lular thiols. CM-H 2DCFDA requires an additional oxi-

ation step before becoming fluorescent. This probecan be used for following stimulation of oxidativeact v ty y externa agents or natura er ce s over anextended period of time Chun et al. , 2001; Kirklan et al. , 2002 . An a quot of fres me a was a e at 10µM f na concentrat on of CM-H 2DCFDA to a 96-we

p ate, an ce s were ncu ate at 37°C 5% CO for 30to 60 min. After removing media containing CM-H2DCFDA, cells were carefully washed with 1X PBS pH 7.2 to remove any excess CM-H D FD .Subsequently, cell were treated with DA or MPP witand without experimental drugs, and at different time

points the absorbance was read at 485/535nm, 1.0s,using the Victor Wallace multi well assay plate reader.A stan ar curve generate from nown concentrat onso H 2O2 was utilized to determine the sensitivity anre a ty of t e assay. T e ata were t en tota e ,

averaged and compared to the 0.0 mM DA or MPP+

treatment condition.

ImmunocytochemistrySH-SY5Y cells were seeded at 5 x 10 4 cells cm 2 n

poly- D-lysine (Sigma)-coated glass coverslips (Fisher Scientific). Cultures were differentiated using either RA or RA/TPA differentiation protocols as describeda ove. Fo ow ng fferent at on, cu tures were f xewith 4% paraformaldehyde for 1 h at room temperature


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.X-100 in TBS 25 mM Tris, pH 7.4; 140 mM NaCl; 3mM KCl). Cultures were incubated in blocking buffer TBS, 2% norma goat serum, 5% BSA, 0.1% Tween-

20) for 1 h at RT then placed in a 1 µg/ml dilution of ant -m crotu u e assoc ate prote n-2 MAP-2, SantaCruz overn g t at 4°C. Fo ow ng ncu at on, t e cu -tures were rinsed with TBST TBS, 0.1% Tween-20s x t mes, at 5 m n eac r nse, an t en p ace n sec-on ary ant o y A exa 568 ant -ra t IgG, 1 µg m ,Molecular Probes) for 2 h at RT. Cultures were againrinsed with TBST (six times, 5 min each) and thenncu ate overn g t at 4°C n a 5 µg m ut on of one

of the following primary antibodies: anti-tyrosiney roxy ase TH, C em con AB152 , ant - opam ne

transporter (DAT, Chemicon MAB369), anti-vesicular monoamine transporter VMAT-2, Chemicon AB1598P , ant -DA D 2 receptor D 2R, C em conAB1558 or anti-DA D receptor D R, Chemicon

AB1785P). Following incubation, the cultures wererinsed with TBST six times, 5 min each and then

placed in appropriate secondary antibody (Alexa 488ant -ra t or ant -rat IgG, 1 µg m , Mo ecu ar Pro esfor 2 h at RT. Cultures were rinsed with TBST sixt mes, 5 m n eac , mounte on g ass s es F s er Sc ent f c w t S ow Fa e L g t Mo ecu ar Pro esand visualized using Olympus Optical's ConfocaLaser Scann ng M croscopy System mo e IX70 w tthe Flouview program (version 2.1.34). Appropriatecontrols were run in parallel such as omission of the

primary antibody.

Western BlottingSH-SY5Y cells were grown in T-25 flasks to 50% con-f uency an fferent ate w t RA an TPA as

escribed above. Cells were removed from the flasksan was e 3 t mes w t PBS uffer pH 7.4 . T eresu t ng ce pe ets were store at -80°C. To extractthe protein, cells were incubated on ice for 15 min incell lysis buffer (10 mM Tris-buffer, 5 mM EDTA, 150mM NaC , 0.5% Tr ton X-100 v v w t Comp ete

protease inhibitor (Roche Diagnostics GmbH;

Mann e m, Germany an t en centr fuge at 16000 g for 20 min at 4°C. The supernatant was collected and

protein concentration determined using Bio-Rad pro-te n assay reagent. T e prote n was oa e at 20 µg of

prote n per we an separate on a 4% 10% SDS-po y-acrylamide gels and then transferred to PVDF mem-

rane Immo on-P: M pore Corporat on, Be for ,MA, USA . After a 1 oc n B oc ng Reagent TBS(AMRESCO; Solon, OH, USA), the membranes werencu ate w t pr mary ant o y 1:1000 n TBST

- .4°C, rinsed 5 times in fresh TBST and incubated for 8h at 4°C in horseradish peroxidase- (HRP-) secondaryant o y 1:10000 n TBST uffer, r nse 5 t mes nfresh TBST and visualized. EnhancedChemiluminescence (Supersignal: Pierce; Rockford,IL, USA was use to v sua ze t e an s on t e mem-

brane. Lane Analysis (densitometry) was performeusing Imaging Research Incorporated (St. Catharines,ON, Cana a AIS 6.0.

Filtration Assay for Inhibition of [ H] DA and [ H]MPP + Uptake in SynaptosomesC57b mice (28 g) and Sprague-Dawley rats (10 weeksage were purc ase from Jac son La orator es Bar Harbor, ME, USA), and housed in a temperature con-trolled room with a 12 h day and night cycle, with freeaccess to foo an water. A an ma s were treate naccor ance w t a protoco approve y t e Sun Hea t

Research Institute Animal Care and Use Committee.T e an ma s were sacr f ce y cerv ca s ocat on ant e r ra ns rap y remove n Art f c a Cere raSpinal Fluid [(ACSF) composed of NaCl (124 mM),KC 4.9 mM , MgSO 4.7H 2O 2.0 mM , CaC 2, 2.0mM , KH PO 4, 1.2 mM , NaHCO , 25.6 mM ang ucose 10.0 mM at pH 7.6] un er 3 m n p ace n50 ml Falcon tubes, and snap frozen in liquid nitrogen.SH-SY5Y ce s were mec an ca y arveste , pe eteat 800 g in 50 ml Falcon tubes, media removed, andsnap frozen in liquid nitrogen. All samples were storedat -80°C until the uptake assay was performed.

Synaptosoma preparat on an upta e assays werecompleted according to methods described byEs eman an assoc ates Es eman et al. , 2001 .Samp es were p ace on ce unt t awe ; t e cau ate-

putamen was dissected and placed in ACSF 10 timest e t ssue vo ume an SH-SY5Y ce s were a so

p ace n ACSF 10 t mes t e pe et vo ume . T e samples were homogenized using a hand-held tissuehomogenizer (Kontes, Vineland, NJ, USA). The

omogenates were centr fuge at 1000 g or 10 m n at4°C; the supernatant was decanted and centrifuged at

20,000 g or 20 m n at 4°C. T e pe et was re-suspen -ed in 10 times the pellet volume of ACSF and assayedfor protein concentration using the Bio-Rad protein

.An a quot of t e synaptosoma preparat on, 50 µ

(final protein concentration of 20 µg), was pre-incubat-e w t or w t out maz n o 5 µM for 20 m n n 4-mtu es F s er Sc ent f c, Tust n, CA, USA at 4°C.Then 200 µl of ACSF solution containing 100 nM buta-c amo oc ng [ 3H]DA or [ H]MPP + rom n ng to


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1 2 +

NEN, Boston, MA, USA or [ H]DA Amers am,UK) was added and then placed in a 25°C waterbathfor 10 min. Specific uptake was defined as the differ-ence n upta e o serve n t e presence an a sence of maz n o 5 µM . Upta e was term nate after 10 m n

by dilution in 3 ml of ice cold ACSF and filtration by aBran e arvester t roug W atman GF C f ters pre-soa e n 0.05% po yet y en m ne. Sc nt at on f uwas added to each individual filter in a scintillation vialan ra oact v ty measure us ng a Wa ac 1411 quscintillation counter. Each experiment contained atleast an n=3 with triplicate determinations. Uptake datawere analyzed by nonlinear regression using GraphpadPr sm 3.0.

Statistical AnalysisData are represente as mean ± S.D. mean ± S.E.M. nthe uptake studies . Unless otherwise noted, differ-

ences were teste for s gn f cance us ng two-wayana ys s of var ance ANOVA fo owe yBonferonni's post-hoc analysis. A P value less than0.05 denoted statistical significance. For some experi-ments fferences etween groups were teste for s g-nificance using One-way ANOVA followed byBonferonn 's post-hoc ana ys s.

RESULTS

Phenotypic Characteristics of DifferentiationSH-SY5Y ce s fferent ate w t RA, TPA or RA TPA un erwent o v ous morp o og ca c angesduring the 6-day period. The cells stopped replicatingan ecame a sta e popu at on. Un fferent ate ce scontinued mitosis and had few if any short processes.The RA and the TPA differentiated cells exhibited aclear neuronal morphology with elongated processesexten ng from oppos ng en s. T e ce s typ ca y werearranged in small clusters with processes extending

etween t em. T e RA TPA fferent ate ce s weret e most ramat ca y c ange ; t e ce s were fre-

quently concentrated in large clusters with numerouse ongate processes connect ng t e c usters, resem- bling explant cultures. The RA/TPA differentiated cellswere immunopositive for TH, D 2 an D receptor, DATand VMAT (FIG. 1). In these cells grown at very low

ensity, DAT immunoreacitivty is observed in the processes of cells (FIG. 3-B) and at lower density than

n cells grown at higher density. When grown at thedensity used in Western blotting (see below) and neu-rotox c ty exper ments, t e eve s of mmunoreact v ty

proximal portion of the processes.

In contrast to t e RA TPA fferent ate ce s, RA f-ferentiated SH-SY5Y cells (FIG. 2) show negligiblemmunoreactivity against antibodies for TH, DAT an

D receptor, ut a g VMAT mmunoreact v ty.This is consistent with evidence that PC-12 cells dif-ferent ate w t RA ex t a c o nerg c p enotypeand decreased expression of dopaminergic markersMatsuoka et al. , 1989), but SH-SY5Y cells terminallyfferent ate w t RA fo owe y TPA ex t an

enhancement of the dopaminergic phenotype(Pennypacker et al. , 1989). To quantify levels oexpression of these proteins after differentiation, west-ern blotting for anti-TH, anti-DAT, anti-VMAT wasutilized. Six-day differentiation with TPA/TPA or witRA TPA ncrease t e amount of TH y 3-fo anDAT by 4-fold as compared to undifferentiated cells or RA treatments alone FIG. 3-A, 3-B . In contrast,

TPA TPA fferent at on an RA TPA fferent atecells show significantly lower levels of VMAT as com-

pared to undifferentiated cells or RA treatment alone(FIG. 3-C). As compared to UN cells the RA, TPA andRA/TPA had significant but relatively small increasesin ß-actin and MAP-2 expression (FIG. 4-F, 4-G), indi-cat ng t at ncrease eve s of TH an DAT were notsimply due to increased neuronal growth induced byTPA/TPA or RA/TPA. While protein levels for the Dand D 3 receptor were almost undetectable in UN cells,t e fferent at on con t ons s gn f cant y a tere t elevels of expression (FIG. 3-D, 3-E). RA, TPA andRA/TPA treatment elevated protein levels for the Dreceptor, and TPA/TPA differentiation and RA/TPA

ifferentiation increased the amount of D receptor by3 and 6-fold, respectively (FIG. 3-E).

Kinetics of Uptake of [ 3H]DA and [ 3H]MPP inRA/TPA and RA/RA Differentiated CellsTo determine if the high levels of DAT in the RA/TPAtreated cells were functionally similar to that in vivo ,we compare t e net cs of [ H]DA an [ H]MPP +

uptake in RA/TPA differentiated cells as comparedwith that of mouse and rat synaptosomal preparationsof cau ate-putamen conta n ng DA term na s. In pre-liminary experiments, we optimized the assay condi-tions for specific [ H]DA and [ H]MPP uptake to DATn mouse, rat an SH-SY5Y synaptosomes. Max ma

uptake rates were determined for all groups (FIG. 4-A,4-B, 4-C ; the V ma values for [ H]DAuptake for mouse(36.69 ± 15.4 pmol/mg protein) and rat (32.82 ± 5.8

pmol/mg protein) were almost identical and similar tothat reported by Eshleman and associates (Eshleman et


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FIGURE 1 Immunocytochemical detection of dopaminergic markers in RA/TPA differentiated SH-SY5Y Cells. Immunocytochemistry andconfocal laser scanning microscopy were as described in Materials and Methods.Panel A is RA/TPA cells incubated with rabbit anti-tyrosine hydroxylase polyclonal antibody.Panel B is RA/TPA cells incubated rat anti-dopamine transporter monoclonal antibody.Panel C is RA/TPA cells incubated with rabbit anti-vesicular monoamine transporter 2 polyclonal antibody.Panel D is RA/TPA cells incubated with rabbit anti-dopamine D receptor polyclonal antibody.Panel E is RA/TPA cells incubated with rabbit anti-dopamine D 3 receptor polyclonal antibody.Panels A 1 E 1 are RA/TPA cells incubated with mouse anti-MAP-2 monoclonal antibody.

Note that as compared to RA differentiated cells (FIG. 2) there are higher levels of TH ( A DAT ( B) but lower levels of VMAT ( C ). Levelsof D 2 (D) and D ( E ) receptor levels also are higher in RA/TPA differentiated cells.


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al. , 2001 . T e K values were also similar to thatreported by Eshleman and associates (Eshleman et al. ,2001) and not significantly different in mouse and ratcau ate-putamen preparat ons. T e V max va ue for [ H]DA uptake in RA/TPA treated SH-SY5Y cells(33.69 ± 9.5 pmol/mg protein) was also very similar tot at er ve from mouse an rat cau ate-putamen

preparat ons ut t e K was higher. Direct comparisonof V max and K m values in RA/TPA treated cells withRA RA treate ce s FIG. 4-B s owe t at t e V maxwas significantly higher in the RA/TPA treated cells ( t -test, P


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than for [ 3H]DA uptake, they were not statistically dif-ferent.

MPP + and DA NeurotoxicityIt as een propose t at t e re at ve eve s of expres-s on of DAT an VMAT cr t ca y regu ate t e sens t v-ty of DAergic neurons to MPP + Przedborski et al.

2000 in vivo . To test furt er f t e re at ve eve s oDAT an VMAT n t e SH-SY5Y ce s un er vary ngdifferentiation conditions altered their sensitivity toMPP + n RA as compare to RA TPA fferent atecells, the cytotoxicity of MPP + was assayed over a four-

ay period at 24-h intervals, utilizing sister cultures int e MTT assay t at measures m toc on r a funct on(FIG. 5-A, 5-B, 5-C). There were significant treatment(cell type, P


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varying concentrations of DA (0 to 500 µM) wereadded to the media and at 24 h intervals the cell viabil-ity was determined in sister cultures utilizing cellcounts, LDH re ease an MTT assay FIG. 6 . T erewere significant treatment (concentration, P


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FIGURE 5 Neurotoxic effects of MPP+ ( A-C ) and DA ( D, E in RA and RA/TPA differentiated SH-SY5Y cells. SH-SY5Y cells weretreated with different concentrations of MPP (0, 0.03, 0.3 and 3.0 mM) or DA (0 to 500 µM) after 6 days of differentiation by retinoic acid(RA), or RA plus TPA (RA/TPA) and the neurotoxic effects of MPP + monitored at 24, 48, 72 and 96 h by MTT assay ( A, B, C or LDHrelease ( D, E ). RA cells were resistant to MPP + at all but the highest concentration of MPP +. In contrast, the RA/TPA differentiated cellsexhibited pronounced effects at the lowest concentration of MPP by 24 h post-MPP + and maintaining the sensitivity at 72 and 96 h ( A). Atthe higher concentrations the differences in sensitivity between differentiation conditions existed at 24 and 48 h but not 72 and 96 h post-MPTP. The cytotoxic effects of DA existed at concentrations of 150 µM and greater but did not differ in sensitivity between RA and RA/TPAdifferentiated cells. The values are the mean ± standard deviation ( n= 3), two-way ANOVA showed significant treatment (cell type, P


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Effect of Transporter Inhibiitors on NeurotoxicityInduced by MPP and DATo determine if MPP + toxicity required intracellular accumulation via DAT, as it does in vivo (Gainetdinovet al. , 1997 , RA RA an RA TPA fferent ate ce swere treated concurrently with the selective DAT

blocker GBR 12909 Matecka et al. , 1996 in the pres-ence of fferent concentrat ons of MPP +. T ere weresignificant treatment (cell type, P


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(FIG. 10-A) or mitochondrial dysfunction (FIG. 10-B)as a measure of neurotoxicity. At the concentration of

pramipexole utilized, the neurotoxic effects of the lowconcentrat on of MPP + was re uce y more t an 50%and was significant even at the highest concentration oMPP +. In contrast, RA treated cells exhibited concen-trat on- epen ent ce eat t at was not attenuate y

pretreatment with PPX.To determine if pretreatment was necessary for the

neuroprotective effects of pramipexole RA and

RA/TPA differentiated cells were treated concurrentlyor 72 h prior to MPP . There were significant treatment6 groups, P


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presence or absence of a D /D 3/D4 antagonist (spiper-one , D 3 preferring antagonist (U99194A), or the D 1receptor antagon st SCH 23390 FIG. 11-B . T erewere significant treatment (drug, P


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DISCUSSION

SK-N-SH cell line, established in the early 1970s, waser ve from ma gnant tumors of mmature neurons

(Ross et al. , 1983) and has been shown to exhibit prop-ert es of stem ce s, cons st ng of a eterogeneous pop-u at on of ce types t at nterconvert. W e SK-N-SHcells express enzymes for DA and NE synthesisOyarce an F em ng, 1991 , t ey ave een reporte

to secrete more DA t an NE in vitro R c ar s anSadee, 1986 . These cells have since been cloned tocreate the line known as SH-SY5Y, which maintainsstem ce c aracter st cs Ross et al. , 1983 an s capa-

ble of proliferating in culture for long periods withoutcontam nat on B e er et al. , 1978; Ross et al. , 1983;Willets et al. , 1995). These cells have been utilized tostudy mechanisms of apoptosis by MPP and neuropro-tect on y DA agon sts Fang et al. , 1995; S ee an et al. , 1997; Song et al. , 1997; Kitamura et al. , 1998 .

Apoptosis produced by MPP+

is correlate with forma-t on of ROS n t e m toc on r a, srupte e ectrontransport, collapse of the mitochondrial potential,release of cytochrome from the organellar to thecytoso c fract on, fo owe y DNA a er ng I tanoand Nomura, 1995; Fall and Bennett, Jr. 1999; Veech et al. , 2000; Kitamura et al. , 1998; Kakimura et al. ,2001). Concurrent treatment with pramipexoleCassarino et al. , 1998) or pretreatment with DA ago-

nists bromocriptine, talipexole and pramipexole for 4ays s gn f cant y re uce MPP + neurotox c ty to t e

neuroblastoma SH-SY5Y cell line Kitamura et al.1998). A high concentration of pramipexole reducet e eve s of ROS cause y MPP + an sta ze t emitochondrial transition pore (Cassarino et al. , 1998).Pretreatment for 4 days with pramipexole or talipexolewas shown to increase levels of the anti-apoptotic pro-tein Bcl-2 and Bcl-X , prevent the translocation ocytochrome from the organellar to the cytosolic frac-


FIGURE 10 Pramipexole pretreatment antagonizes the effects of MPP + in RA/TPA but not RA differentiated SH-SY5Y cells. RA andRA/TPA differentiated SH-SY5Y cells were treated with different concentrations of MPP (0, 0.03, 0.3 and 3.0 mM) 3 days after exposure(A, B ) or simultaneous ( C with pramipexole (PPX, 1.0 mM) or vehicle (VEH) and the neurotoxic effects of MPP + monitored at 48 h by

LDH assay ( A) and MTT assay ( B, C ). Note that the neurotoxic effects of MPP+

were greater in the RA/TPA as compared to the RA dif-ferentiated cells at each concentration of MPP +. For A and B, note that the neurotoxic effects of MPP + were significantly antagonized by pretreatment with pramipexole in the RA/TPA but not the RA differentiated cells. Similar results were determined by MTT and LDH assays.The values are the mean ± standard deviation ( n=3), two-way ANOVA showed significant treatment (cell type, P


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tion produced by MPP +, inhibit cytochrome c activa-t on of caspase-9, an prevent DNA a er ng pro-

uce y MPP + K tamura et al. , 1998; Ka mura et al.2001 . D 2-selective and non-selective antagonists did not , owever, n t t e neuroprotect ve effects of ta - pexole or pramipexole against MPP +-induced apopto-sis of SH-SY5Y cells Kitamura et al. , 1998 , and free

ra ca scaveng ng propert es of t ese compoun s ave been favored as the mechanism of neuroprotection.This might invalidate the use of SH-SY5Y cells tostu y t e neuroprotect ve effects of DA agon sts, s nceDA receptor effects in vivo do utilize DA receptor me iate effects.

There are problems with use of the undifferentiatedSH-SY5Y cells as a model for dopaminergic neuronsas there is not high expression of DA syntheticenzymes, tox c ty y MPP oes not requ re DAT, anongoing mitosis can dramatically affect the response to

rugs an tox ns. It a een prev ous y reporte t at

RA fferent at on of SH-SY5Y ce s fo owe y treat-ment with the phorbol ester TPA significantly elevates

pro uct on of DA synt et c enzymes Pennypac er et al. , 1989), D receptors (Farooqui 1994) and its G-pro-te ns Ammer an Sc u z 1994 . We conf rme t s yidentifying that SH-SY5Y cells were immunoreactiveto TH and DAT antibodies, and that membranes pre-

pared from the SH-SY5Y cells were also immunoposi-t ve us ng western ott ng. Term na fferent at onwith RA followed by TPA treatment significantlyncreased the amount of TH and DAT as compared to

UN, or RA fferent ate ce s. In contrast, com netreatment with RA and TPA actually reduced levels oVMAT. To test the appropriateness of the RA/TPA dif-ferent ate ce s as a opam nerg c mo e ce ne wefurther determined that the RA/TPA cells were sensi-tive to concentrations of MPP + lower than that requiredfor un fferent ate SH-SY5Y ce s a ove 1 mM(Fall and Bennett, Jr., 1999)), and that treatment of ce s w t t e DAT antagon st GBR 12909 resu te nan a most comp ete oc a e of t e tox c ty of MPP +


FIGURE 11 DA receptor mediated effects of pramipexole against MPP + (A, B ) and DA ( C ). A . Effect of various concentrations of pramipexole on MPP + induced neurotoxicity. RA/TPA differentiated SH-SY5Y cells were treated with MPP + (0 and 0.1 mM) 3 days after exposure to varying concentrations of pramipexole (PPX, 0.0 to 1.0 mM) and the neurotoxic effects of MPP + monitored at 48 h by MTTassay. The values are expressed as percentage of the MTT inhibition in cells incubated with MPP + nly and are the mean ± standard devia-tion ( n=3). Significance protection from MPP + t * P


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This is in contrast to undifferentiated SH-SY5Y cellsFang et al. , 1995; W ets et al. , 1995 , ut s m ar to

w at occurs n pr mary cu tures of mesencep a copaminergic neurons (Bilsland et al. , 2002 .

Furt ermore, we foun t at RA TPA fferent ate SH-SY5Y ce s ex te spec f c DAT me ate DA an

+ uptake with very similar kinetics to mouse anrat str ata synaptosomes conta n ng DA term na s. Our resu ts w t mouse an rat str ata synaptosomes aresimilar to that reported by Eshleman and associatesEs eman et al. , 2001 , an t e compara ty etween

the RA/TPA differentiated SH-SY5Y cells and mouseand rat striatal synaptosomes is indicative that thesece s ex t g nat ve express on of a funct onaorm o DAT. T e g er K in the cells as compared

to the mouse and rat striatal synaptosome likely is dueto t e a sence of catec o - O-met y tranferease n t ecells, which is known to alter the kinetics of DA trans-

port Es eman et al. , 1997 . In contrast to t e RA TPA

fferent ate ce s, RA RA fferent ate ce s ex t-ed significantly less DA uptake, were less sensitive to

+- n uce tox c ty, an t e MPP + tox c ty was notinhibited by a DAT blocker. This is similar to undiffer-entiated SH-SY5Y cells in which inhibition of cate-cholamine uptake does not block MPP +-induced neuro-tox c ty, n cat ve of w y suc g concentrat ons oMPP + are required to induce apoptosis (Fang et al. ,1995; W ets et al. , 1995 . Our ata support t e pro-

posa t at t e rat o of DAT to VMAT can mo u ate t esensitivity of SH-SY5Y cells to MPP +, as it does in vivoGa net nov et al. , 1997 . T us, RA TPA fferent at-

ed SH-SY5Y cells exhibit characteristics consistentwith cultured DA neurons, but represent a more feasi-

ble model to explore the mechanisms of cell death pro-uced by MPP +.While DA agonist mechanisms providing neuropro-

tect on are var e an pro a y nc u e t e r free-ra -cal scavenging properties (Cassarino et al. , 1998;Kitamura et al. , 1998; Ling et al. , 1998; Grünblatt et al. , 1999 , t wou e mportant to emonstrate DAreceptor mediated effects in cell culture. Our resultssuggest that the robust expression of D 2 and, particu-

ar y, D 3 receptors n t e RA TPA fferent ate ce s snecessary for DA receptor-mediated protection by

pram pexo e. Cons stent w t w at as een o serven undifferentiated SH-SY5Y cells we determined that

pretreatment of SH-SY5Y ce s w t t e D preferr ngagonist pramipexole for three days before addition of MPP + o the RA TPA ifferentiate cells re uce MPP +

neurotox c ty as measure y t e MTT assay n t eRA/TPA differentiated cells. While a similar degree of

protection was measured by LDH release, the degree of

cell loss measured with this protocol is less than thatw t MTT at t e same t me po nts. Ce s w t stur em toc on r a funct on, as eterm ne y t e MTTassay, may not be exhibiting cell lysis or membrane

srupt on, as measure y t e LDH re ease assay, untater t me po nts. Important y, concurrent treatment

with pramipexole did not afford protection to the neu-rotox c effects of MPP + n t e RA TPAtreate ce s anne t er pretreatment nor concurrent treatment waseffective in RA differentiated cells. The RA differenti-ate ce s w t ow express on of D 2 an , part cu ar y,D3 receptors exhibited negligible protection by

pram pexo e. We emonstrate concentrat on- epen -ent effects for neuroprotection by pramipexole, consis-tent w t receptor me ate effects, an t at a D 2 D3 D4or D 3 preferring receptor blocker potently antagonizet e neuroprotect ve effects of pram pexo e. In contrast,the D 1 receptor antagonist SCH 23390 was ineffective.T us, t s un e y t at t e neuroprotect ve effects areso e y epen ent on t e ant ox ant capa t es o

pramipexole and support evidence by Carvey and Ling1997 , t at at east some of t e neuroprotect ve effects

of pram pexo e are D 3 receptor me ate . T e co-express on of D 2 an D receptors n t e RA TPA f-ferentiated cells is phenotypically similar what exists inthe brain, in which DAergic neurons express D an Dreceptors Le Moine and Bloch 1991; Diaz et al. , 2000and both of which are functional Gobert et al. , 1995;Koe tzow et al. , 1998; L' ron e et al. , 1998;Dickinson et al. , 1999; Jung et al. , 1999; Zapata et al. ,2001; Zapata and Shippenberg, 2002). Hence, termi-nally differentiated SH-SY5Y cells make a good modelfor what occurs in vivo , and the role of DA receptor-mediated neuroprotection against MPP can be morec ear y ef ne .

Our data also indicate that DA-induced cell death andthat by MPP n the RA TPA ifferentiate cells occursv a fferent mec an sms. It as een prev ous y s ownn a variety of cell lines that excess DA added to the

media will produce cell death associated with depletionof GSH an an ntrace u ar r se n ROS Masserano et

al. , 1996; Lai and Yu 1997; Jones et al. , 2000; Stokeset al. , 2000). We show here that excess DA added to theme a ncreases t e format on of H 2O an re uct on oGSH, and a concomitant increase in intracellular ROSan re uct on of GSH. In contrast, MPP + oes not pro-duce an intracellular rise in ROS, nor was it additive tothe ROS effects of DA. Interestingly, neither the celdeath nor rise in intracellular ROS is affected by block-ade of DA, NE, 5-HT or a combination of blockers ouptake. This is inconsistent with some studies



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