Terminally Differentiated SH-SY5Y Cells Provide A Model (887035)

Neurotoxicity Research, 2004, VOL. 5(8). pp.
579-598
F.P. G
Graham
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Publishing
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Terminally Differentiated SH-SY5Y Cells Provide a Model

System for Studying Neuroprotective Effects of Dopamine
Agonists
STEVEN P. PRESGRAVESa, TARIQ AHMEDb, SABINE BORWEGEb and JEFFREY N. JOYCEb,*
aMolecular and Cellular Biology Graduate Group, Arizona State University, Tempe, AZ; and bThomas H. Christopher
Center for Parkinson's Disease Research Center, Sun Health Research Institute, 10515 West Santa Fe Dr., Sun City; AZ
85351. Jeff.Joyce@sunhealth.org
(Received 28 January 2004; Revised 27 February 2004; In final form 27 February 2004)
We characterized undifferentiated (UN) and three

differentiation conditions of the SH-SY5Y neuroblastoma cell line for phenotypic markers of
dopaminergic cells, sensitivity to the neurotoxin 1methyl-4-phenyl-1,2,3,6-tetrahydropyridinium ion
(MPP+), the requirement to utilize the dopamine
(DA) transporter (DAT) for MPP+ toxicity, and the
neuroprotective effects of pramipexole. Cells were
differentiated with retinoic acid (RA), 12-O-tetradecanoyl-phorbol-13-acetate (TPA), and RA followed
by TPA (RA/TPA). RA/TPA treated cells exhibited
the highest levels of tyrosine hydroxylase and DAT
but lower levels of vesicular monoamine transporter. The kinetics of [3H]DA uptake and [3H]MPP+
uptake to DAT in RA/TPA differentiated cells were
similar to that of rat and mouse caudate-putamen
synaptosomes. RA/TPA differentiated cells evidenced high sensitivity to the neurotoxic effects of
MPP+ (0.03 to 3.0 mM), and the neurotoxic effects of
MPP+ were blocked with the DAT inhibitor 1-(2[bis(4-fluorophenyl)methoxy]ethyl)-4-(3-phenylpropyl)piperazine (GBR 12909). DA-induced cell
death was not more sensitive in RA vs RA/TPA differentiated cells and was not inhibited by transporter inhibitors. RA/TPA differentiated cells
exhibited 3- fold and 6-fold higher levels, respectively, of DA D2 and D3 receptors than UN or RA differentiated cells. Pretreatment with pramipexole
was protective against MPP+ in the RA/TPA differentiated cells but not in undifferentiated or RA differentiated cells. The neuroprotective effect of
pramipexole was concentration-dependent and
dopamine D2/D3 receptor dependent. In contrast,
protection by pramipexole against DA was not DA
receptor dependent. Further characterization of the
neuroprotective effects of DA agonists in this model

system can provide unique information about DA
receptor dependent and independent mechanisms
of neuroprotection.
Keywords: Parkinson's disease; D2 Receptor; D3 Receptor;
MPP+; Dopamine transporter; Pramipexole
INTRODUCTION
Parkinson's disease (PD) is a neurodegenerative disorder with an insidious onset and a prolonged course over
many years. The primary cause of the symptoms of this
illness is the death of dopamine- (DA-) producing neurons of the substantia nigra (SN) and the resultant
depletion of DA in the striatum (Hornykiewicz, 1998).
While L-3,4-dihydroxyphenylalanine (L-dopa) provides effective symptomatic treatment for PD, it does
not alter the progression of the disease. DA agonists are
also used for treatment of PD, some of which have
been shown to be neuroprotective in Parkinson's models (Anderson et al., 2001) and, perhaps, in PD itselff
(Marek et al., 2002). Antiparkinsonian agents that are
direct DA agonists such as apomorphine (Grnblatt et
al., 1999), bromocriptine (Muralikrishnan and
Mohanakumar, 1998), and pramipexole (Kitamura et
al., 1997), have been shown to be neuroprotective
against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP-) induced damage to the DA system in mice.
MPTP administration to mice is considered a good
model of studying neuroprotection because MPTP is
known to produce parkinsonism in humans and in subhuman species through selective loss of DA-ergic neurons of the SN (Burns et al., 1983; Langston et al.,
*Corresponding author. Tel: +1 623 876-5439; Fax: 1 623 876-5695; E-mail: Jeff.Joyce@sunhealth.org
ISSN 1029 8428 print/ ISSN 1476-3524 online. 2004 FP Graham Publishing Co., www.fpgrahamco.com
580
S.P. PRESGRAVES et al.
1983). There are a number of related compounds to

1983)
MPTP that produce nigral cell loss in primates
(McNaught et al., 1996). MPTP produces apoptotic
changes associated with PD ( Cohen and Werner, 1994;
Cassarino and Bennett Jr., 1999; Hirsch et al., 2000);
and MPTP produces ongoing cell death in humans for
decades after the initial insult (Langston et al., 1999).
Hence, drugs that reduce the neurotoxicity of compounds such as MPTP, or its active metabolite MPP+,
may prove to be neuroprotective in PD.
However, the efficacy of current DA agonists in neuroprotection is less than what is desirable and an understanding of the mechanisms of protection might provide better targets. Because the DA D3 receptor preferring agonists pramipexole and ropinirole appear to be
the most potent of the agonists in vivo (Kitamura et al.,
1997; Iida et al., 1999; Zou et al., 2000) it has suggested that the D3 receptor plays an important role. This has
been further substantiated by the findings that the neuroprotective effects of pramipexole against MPTP is
reduced in D3 receptor knockout mice (Ramirez et al.,
2003). Cell cultures have the advantage of identifying
even more specifically the receptor and nonreceptor
mediated effects of drugs and it has been establised that
pramipexole can prevent neurotoxicity produced by Ldopa in mesencephalic cultures (Carvey et al., 1997;
Ling et al., 1999) and by MPP+ in the neuroblastoma
SH-SY5Y cell line (Kitamura et al., 1998). Carvey and
associates have provided valuable evidence the D3
receptor mediates a major component of the effects of
pramipexole (Carvey et al., 2001). In contrast, another
group has shown that blockade of DA receptors with
either D2-selective or non-selective compounds did not
inhibit the neuroprotective effects of talipexole or
pramipexole against MPP+-induced apoptosis of SHSY5Y cells (Kitamura et al., 1998). This apparent conflict might reflect differences in the cell culture system
used, or the toxin utilized.
While mesencephalic cultures have the advantage of
being a source of DA neurons, L-dopa induced neurotoxicity may not utilize the same cell death pathways as
MPP+ (Murer et al., 1998; Choi et al., 1999; Koshimura
et al., 2000; Blum et al., 2001). MPP+, which has been
used in the SH-SY5Y cells, does mimic many aspects
of the DA neuron death observed in PD. The human
neuroblastoma cell line SH-SY5Y, which was subcloned from the SK-N-SH cell line; exhibits neuronal
properties and a catecholaminergic phenotype including the ability to transport DA and NE, express
enzymes for the synthesis and metabolism of DA and
acetylcholine, and express receptors for DA and acetylcholine (Biedler et al., 1978; Ross et al., 1983); Willets
et al.
al , 1995).
1995) Treatment of the SH-SY5Y
SH SY5Y cells with
+
high concentrations of MPP for 3 days will produce
cell death with clear morphological evidence of apoptosis (Fang et al., 1995; Sheehan et al., 1997; Song et
al., 1997; Kitamura et al., 1998). However, the concentrations of MPP+ that are required to produce neurotoxicity in the undifferentiated state of this cell line
are higher than those needed for mesencephalic cultures (Storch et al., 2000a). While it has been assumed
that 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;
Kitamura et al., 1998), as it does in vivo, the relatively
high resistance of these cells to this compound would
suggest otherwise. The requirement to use relatively
high concentrations of MPP+ to produce neurotoxicity
may be due to the low expression of the DAT and high
expression of vesicular monoamine transporter
(VMAT) in this cell line in the undifferentiated state.
To test if the insensitivity of the neuroblastoma SHSY5Y cell line to MPP+ and to the DA receptor-mediated effects of pramipexole reflected properties of the
undifferentiated SH-SY5Y cells, we examined this cell
line after treatment with factors that differentiate these
cells to a phenotypically mature dopaminergic cell type
(Pennypacker et al., 1989). We compared the neurotoxic effects of MPP+ with that of DA which is thought
to produce nonapoptotoic death (Gmez-Santos et al.,
2003). We have identified that SH-SY5Y cells differentiated with retinoic acid (RA) and the phorbol ester
12-O-tetradecanoyl-phorbol-13-acetate (TPA) exhibit
the most pronounced dopaminergic phenotype, characteristics of MPP+-induced toxicity similar to DA neurons in vivo, and D3-like receptor dependent neuroprotection against MPP+.
MATERIALS AND METHODS

Drugs
S(-)pramipexole was provided by Pharmacia
Corporation (Kalamazoo, MI, USA). MPP+ iodide was
obtained from Research Biochemicals (Natick, MA,
USA). All other chemicals were purchase from Sigma
Chemical Co. (St Louis, MO, USA) unless otherwise
stated.
Cell Culture
Human SH-SY5Y neuroblastoma cells (ATCC,
Manassas, VA, USA) were grown to confluence in SHSY5Y media [Dulbecco's Modified Eagle's media
(DMEM) supplemented with 10% fetal calf serum, 100
SH-SY5Y CELLS AS A MODEL TO STUDY NEUROPROTECTION
581
g/ml penicillin,
penicillin 100 g/ml streptomycin,
streptomycin 0.25
0 25 g/ml
amphotericin B (Gibco Grand Island, NY, USA), and
0.01 M non-essential amino acids (Gibco)] and then
sub-cultured for differentiation in 48 well culture plates
(Corning Costar, Corning, NY, USA). Four distinct differentiation protocols were utilized: Undifferentiated
cells (UN) were grown in SH-SY5Y media for 3 days;
then media was removed and replaced with fresh SHSY5Y media for another 3 days growth. Retinoic acid
(RA) differentiated cells were grown in SH-SY5Y
media containing 10 M RA for 3 days; then the media
was removed and replaced with fresh RA media for
another 3 days of differentiation. Phorbol ester differentiated cells were grown in 80 nM 12-O-tetradecanoyl-phorbol-13-acetate (TPA) for 3 days; then the
media was removed and replaced with fresh TPA media
for another 3 days of differentiation. RA/TPA differentiated cells were grown in RA media for 3 days; then
the media was removed and replaced with fresh TPA
media for another 3 days of differentiation.
Pramipexole Treatment
Differentiated (RA and RA/TPA ) SH-SY5Y cells were
either pretreated with media containing pramipexole
(concentration range of 0-1.0 mM) for 3 days then
treated with MPP+ (100 M containing media; or concurrently treated with media containing pramipexole
(concentration range of 0-1.0 mM) and MPP+ or DA.
SH-SY5Y cells were subcultured and differentiated as
described above. RA/TPA differentiated cells were
either pretreated with media containing 0 M
pramipexole, 100 M pramipexole, 100 M pramipexole plus 10 M spiperone, 100 M pramipexole plus 1
M U99194A [5,6-dimethoxy-2-(di-u-propylamino)
indan], or 100 M pramipexole plus 10 M SCH
23390 [R(+)-3-methyl-7-chloro-8-hydroxy-1-phenyl2,3,4,5-tetrahydro-1H-3-benzazepine] for 3 days, then
treated with MPP+ (300 M) or DA (150 M) containing media. Subsequently cell viability was assayed
using the MTT and LDH assays and cell counts at 24 h
intervals.
Cytotoxic Effects of MPP+ and DA on SH-SY5Y

Cells
SH-SY5Y cells were subcultured into 48 well plates
(25,000 cells/well). The cells were then differentiated
as above for RA differentiated and RA/TPA differentiated cells. Each of these populations was then treated
with different concentrations of MPP+ (0.0, 0.03, 0.3
mM, 1.0, and 3.0 mM) or DA (0, 1, 5, 10, 50, 100, 150,
250 and 500 M) in SH-SY5Y media. To test for cell
viability and other measures, sister cultures were initiated at the same time and condition and stopped at time
point for assessments. Cell viability was assayed using
the MTT salt assay, LDH assay and cell counts at 24,
48, 72 and 96 h post MPP+.
MTT Assay for Cell Viability

The cytotoxic affect of the MPP+ was assayed over a
four-day period at 24 h intervals using the MTT (3[4,5-Dimethylthiazol]-2,5-diphenyltetrazolium) salt
assay as described by Sigma. MTT stock solution (5
mg/ml) was added to each well at one tenth the total
media volume and left to incubate at 37C for 4 h. At
the end of this incubation, the media was removed and
the converted dye solubilized with acidic isopropanol
(0.1 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 then
totaled, averaged and represented as a percent cell
death as compared to the 0.0 mM MPP+ treatment off
each differentiation condition over the four-day MPP+
treatment.
Transporter Antagonists
Differentiated (RA and RA/TPA ) SH-SY5Y cells were
incubated with 1-(2-[bis(4-fluorophenyl)methoxy]
ethyl)-4-(3-phenylpropyl)piperazine (GBR 12909) in
SH-SY5Y media for 1 h prior to MPP+ administration
and then treated with different concentrations of MPP+
(0 - 3.0 mM) in SH-SY5Y media. Cell viability was
assayed using the MTT and LDH assays. RA/TPA differentiated SH-SY5Y cells were incubated with 10 M
of the DAT inhibitor GBR 12909, the NE transport
inhibitor, nisoxetine, and the serotonin (5-HT) transport inhibitor 6-nitroquipazine or combinations of the
transport inhibitors for 1 h prior to addition of 200 M
DA, and cell viability was measured at 24 h intervals.
Cell viability was assayed with cell count, GSH levels
and hydrogen peroxide (H2O2) levels.
Lactate Dehydrogenase Assay for Cell Viability

The cytotoxic effect of the MPP+ was assayed over a
four-day period at 24 h intervals using Lactate dehydrogenase method (LDH) of activity as a measure off
cell membrane integrity. The Cytotox 96 (Promega)
assessment of cell viability is a quantitative measure off
lactate dehydrogenase, a stable enzyme, released into
the media upon cell death and is measured colorimetrically based on the conversion of lactate to pyruvate in
the presence of NAD+ coupled to the conversion of a
tetrazolium salt into a red formazan product. Aliquots
of 50 l of media from each well were transferred to a
96 well plate. The remaining media was removed, lysis
buffer was added and the plates were incubated for 1 h
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at 37
37C
C. To assay
assay, buffer and substrate were added to
each transfer plate. The plates were maintained in the
dark and incubated at room temperature for 30 min.
The reaction was stopped and the absorbance of the
converted dye measured at 490 nm. The data were
totaled, averaged, and represented as percent cell
death.
Cell Count Assessment of Cell Viability
SH-SY5Y cells were cultured in 48 well culture plates.
After the individual treatments, cells were detached
and stained with trypan blue (final concentration,
0.023% w/v) for 3 min. The viable, non-colored, and
dead, blue-colored, cells were counted from an aliquot
(1 ml) of cell suspension using a hemocytometer under
400x magnification. The data were analyzed as the
ratio of alive to dead cells (% cell survival).
Protocol for Extracellular and Intracellular
Glutathione Levels
The principle of the procedure is based on the oxidation of reduced glutathione (GSH) by 5,5'-dithiobis(2nitrobenzoic acid) [DTNB], to measure the total glutathione content of a biological sample. Solutions of
DA in the cell culture media were prepared with 1 mM
GSH added. At different time points, aliquots were
taken to measure the GSH remaining in the media
(extracellular GSH). DTNB was freshly prepared prior
to use. To aliquot of media (50 l), 50 l of 10 mM
DTNB was added to a 96-well assay plate (6 samples/treatment), incubated for 10-15 min, and
absorbency was read at 405 nm against a DTNB blank
with Victor Wallace plate reader. For intracellular
measures the cells were centrifuged for 10 min to pellet cells, supernatant was removed and discarded, pellets were resuspended with 250 l Tris-EDTA and
transferred to 1.5 ml microcentrifuge tubes. Cells were
lysed by freezing, then 50 l samples were transferred
to 96-well plates (5 samples/treatment) and 50 l of 10
mM DTNB was added, incubated for 10-15 min, and
absorbency was read at 405 nm against a DTNB blank
with Victor Wallace plate reader. A GSH standard concentration curve (0.5, 1, 2.5, 5, 10, 15 and 20 M) was
used as a standard. The data were then totaled, averaged and represented as a percent change from the 0.0
mM DA treatment.
PeroXOquant Quantitative Peroxide Assay
This assay provides a quantitative measure of H2O2
levels based on oxidation of ferrous to ferric ion in the
presence of xylenol orange. Solutions of DA in the cell
culture media were prepared with 1 mM GSH added.
At different time points,

points aliquots were taken to measmeas
ure the H2O2 levels in the media (extracellular H2O2).
The 20 l aliquots of experimental media from treated
cells were transferred to a 96-well assay plate (6 samples/treatment), 200 l of working solution was added
(PeroXOquant Quantitative Peroxide Assay, Pierce
Chemical, Rockford, IL, USA), incubated for 2025min at room temperature, and absorbency read at
560 nm with Victor Wallace plate reader with a standard curve generated from known concentrations off
H2O2. The data were then totaled, averaged and represented as a percent change from the 0.0 mM DA treatment.
Intracellular Reactive Oxygen Species Levels
Using CM-H2DCFDA
CM-H2DCFDA is a cell-permeant indicator for reactive oxygen species (a Thiol-Reactive tracer) that is
nonfluorescent until removal of the acetate groups by
intracellular esterases, and oxidation occurs within the
cell. Cytoplasmic enzymes hydrolyze the acetate
groups from this membrane-permeant probe and the
chloromethyl moieties become conjugated to intracellular thiols. CM-H2DCFDA requires an additional oxidation step before becoming fluorescent. This probe
can be used for following stimulation of oxidative
activity by external agents or natural killer cells over an
extended period of time (Chun et al., 2001; Kirkland et
al., 2002). An aliquot of fresh media was added at 10
M final concentration of CM-H2DCFDA to a 96-well
plate, and cells were incubated at 37C/5% CO2 for 30
to 60 min. After removing media containing CMH2DCFDA, cells were carefully washed with 1X PBS
(pH 7.2) to remove any excess CM-H2DCFDA.
Subsequently, cell were treated with DA or MPP+ with
and 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 standard curve generated from known concentrations
of H2O2 was utilized to determine the sensitivity and
reliability of the assay. The data were then totaled,
averaged and compared to the 0.0 mM DA or MPP+
treatment condition.
Immunocytochemistry
SH-SY5Y cells were seeded at 5 x 104 cells/cm2 on
poly-D-lysine (Sigma)-coated glass coverslips (Fisher
Scientific). Cultures were differentiated using either
RA or RA/TPA differentiation protocols as described
above. Following differentiation, cultures were fixed
with 4% paraformaldehyde for 1 h at room temperature
(RT) then permeabilized by treatment with 0.1%

0 1% Triton
X-100 in TBS (25 mM Tris, pH 7.4; 140 mM NaCl; 3
mM KCl). Cultures were incubated in blocking buffer
(TBS, 2% normal goat serum, 5% BSA, 0.1% Tween20) for 1 h at RT then placed in a 1 g/ml dilution of
anti-microtubule associated protein-2 (MAP-2, Santa
Cruz) overnight at 4C. Following incubation, the cultures were rinsed with TBST (TBS, 0.1% Tween-20)
six times, at 5 min each rinse, and then placed in secondary antibody (Alexa 568 anti-rabbit IgG, 1 g/ml,
Molecular Probes) for 2 h at RT. Cultures were again
rinsed with TBST (six times, 5 min each) and then
incubated overnight at 4C in a 5 g/ml dilution of one
of the following primary antibodies: anti-tyrosine
hydroxylase (TH, Chemicon AB152), anti-dopamine
transporter (DAT, Chemicon MAB369), anti-vesicular
monoamine transporter (VMAT-2, Chemicon AB
1598P), anti-DA D2 receptor (D2R, Chemicon
AB1558) or anti-DA D3 receptor (D3R, Chemicon
AB1785P). Following incubation, the cultures were
rinsed with TBST (six times, 5 min each) and then
placed in appropriate secondary antibody (Alexa 488
anti-rabbit or anti-rat IgG, 1 g/ml, Molecular Probes)
for 2 h at RT. Cultures were rinsed with TBST (six
times, 5 min each), mounted on glass slides (Fisher
Scientific) with Slow Fade Light (Molecular Probes)
and visualized using Olympus Optical's Confocal
Laser Scanning Microscopy System (model IX70) with
the Flouview program (version 2.1.34). Appropriate
controls were run in parallel such as omission of the
primary antibody.
Western Blotting
SH-SY5Y cells were grown in T-25 flasks to 50% confluency and differentiated with RA and TPA as
described above. Cells were removed from the flasks
and washed 3 times with PBS buffer (pH 7.4). The
resulting cell pellets were stored at -80C. To extract
the protein, cells were incubated on ice for 15 min in
cell lysis buffer (10 mM Tris-buffer, 5 mM EDTA, 150
mM NaCl, 0.5% Triton X-100 (v/v) with Complete
protease inhibitor (Roche Diagnostics GmbH;
Mannheim, Germany) and then centrifuged at 16000g
for 20 min at 4C. The supernatant was collected and
protein concentration determined using Bio-Rad protein assay reagent. The protein was loaded at 20 g of
protein per well and separated on a 4%/10% SDS-polyacrylamide gels and then transferred to PVDF membrane (Immobilon-P: Millipore Corporation, Bedford,
MA, USA). After a 1 h block in Blocking Reagent TBS
(AMRESCO; Solon, OH, USA), the membranes were
incubated with primary antibody (1:1000) in TBST
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buffer (TBS buffer with 00.05%

05% Tween-20) overnight at
4C, rinsed 5 times in fresh TBST and incubated for 8
h at 4C in horseradish peroxidase- (HRP-) secondary
antibody (1:10000) in TBST buffer, rinsed 5 times in
fresh
TBST
and
visualized.
Enhanced
Chemiluminescence (Supersignal: Pierce; Rockford,
IL, USA) was used to visualize the bands on the membrane. Lane Analysis (densitometry) was performed
using Imaging Research Incorporated (St. Catharines,
ON, Canada) AIS 6.0.
Filtration Assay for Inhibition of [3H] DA and [3H]
MPP+ Uptake in Synaptosomes
C57b mice (28 g) and Sprague-Dawley rats (10 weeks
age) were purchased from Jackson Laboratories (Bar
Harbor, ME, USA), and housed in a temperature controlled room with a 12 h day and night cycle, with free
access to food and water. All animals were treated in
accordance with a protocol approved by the Sun Health
Research Institute Animal Care and Use Committee.
The animals were sacrificed by cervical dislocation and
their brains rapidly removed in Artificial Cerebral
Spinal Fluid [(ACSF) composed of NaCl (124 mM),
KCl (4.9 mM), MgSO4.7H2O (2.0 mM), CaCl2, (2.0
mM), KH2PO4, (1.2 mM), NaHCO3, (25.6 mM) and
glucose (10.0 mM) at pH 7.6] (under 3 min) placed in
50 ml Falcon tubes, and snap frozen in liquid nitrogen.
SH-SY5Y cells were mechanically harvested, pelleted
at 800g in 50 ml Falcon tubes, media removed, and
snap frozen in liquid nitrogen. All samples were stored
at -80C until the uptake assay was performed.
Synaptosomal preparation and uptake assays were
completed according to methods described by
Eshleman and associates (Eshleman et al., 2001).
Samples were placed on ice until thawed; the caudateputamen was dissected and placed in ACSF (10 times
the tissue volume) and SH-SY5Y cells were also
placed in ACSF (10 times the pellet volume). The samples were homogenized using a hand-held tissue
homogenizer (Kontes, Vineland, NJ, USA). The
homogenates were centrifuged at 1000g for 10 min at
4C; the supernatant was decanted and centrifuged at
20,000g for 20 min at 4C. The pellet was re-suspended in 10 times the pellet volume of ACSF and assayed
for protein concentration using the Bio-Rad protein
assay.
An aliquot of the synaptosomal preparation, 50 l
(final protein concentration of 20 g), was pre-incubated with or without mazindol (5 M) for 20 min in 4-ml
tubes (Fisher Scientific, Tustin, CA, USA) at 4C.
Then 200 l of ACSF solution containing 100 nM butaclamol (blocking [3H]DA or [3H]MPP+ from binding to
584
D1 or D2 receptors) and various concentrations of MPP+

(NEN, Boston, MA, USA) or [3H]DA (Amersham,
UK) was added and then placed in a 25C waterbath
for 10 min. Specific uptake was defined as the difference in uptake observed in the presence and absence of
mazindol (5 M). Uptake was terminated after 10 min
by dilution in 3 ml of ice cold ACSF and filtration by a
Brandel harvester through Whatman GF/C filters presoaked in 0.05% polyethylenimine. Scintillation fluid
was added to each individual filter in a scintillation vial
and radioactivity measured using a Wallac 1411 liquid
scintillation counter. Each experiment contained at
least an n=3 with triplicate determinations. Uptake data
were analyzed by nonlinear regression using Graphpad
Prism 3.0.
Statistical Analysis
Data are represented as mean S.D. (mean S.E.M. in
the uptake studies). Unless otherwise noted, differences were tested for significance using two-way
analysis of variance (ANOVA) followed by
Bonferonni's post-hoc analysis. A P value less than
0.05 denoted statistical significance. For some experiments differences between groups were tested for significance using One-way ANOVA followed by
Bonferonni's post-hoc analysis.
RESULTS
Phenotypic Characteristics of Differentiation
SH-SY5Y cells differentiated with RA, TPA or
RA/TPA underwent obvious morphological changes
during the 6-day period. The cells stopped replicating
and became a stable population. Undifferentiated cells
continued mitosis and had few if any short processes.
The RA and the TPA differentiated cells exhibited a
clear neuronal morphology with elongated processes
extending from opposing ends. The cells typically were
arranged in small clusters with processes extending
between them. The RA/TPA differentiated cells were
the most dramatically changed; the cells were frequently concentrated in large clusters with numerous
elongated processes connecting the clusters, resembling explant cultures. The RA/TPA differentiated cells
were immunopositive for TH, D2 and D3 receptor, DAT
and VMAT (FIG. 1). In these cells grown at very low
density, DAT immunoreacitivty is observed in the
processes of cells (FIG. 3-B) and at lower density than
in cells grown at higher density. When grown at the
density used in Western blotting (see below) and neurotoxicity experiments, the levels of immunoreactivity
are much higher but still predominantly observed in the

proximal portion of the processes.
In contrast to the RA/TPA differentiated cells, RA differentiated SH-SY5Y cells (FIG. 2) show negligible
immunoreactivity against antibodies for TH, DAT and
D3 receptor, but had high VMAT immunoreactivity.
This is consistent with evidence that PC-12 cells differentiated with RA exhibit a cholinergic phenotype
and decreased expression of dopaminergic markers
(Matsuoka et al., 1989), but SH-SY5Y cells terminally
differentiated with RA followed by TPA exhibit an
enhancement of the dopaminergic phenotype
(Pennypacker et al., 1989). To quantify levels off
expression of these proteins after differentiation, western blotting for anti-TH, anti-DAT, anti-VMAT was
utilized. Six-day differentiation with TPA/TPA or with
RA/TPA increased the amount of TH by 3-fold and
DAT by 4-fold as compared to undifferentiated cells or
RA treatments alone (FIG. 3-A, 3-B). In contrast,
TPA/TPA differentiation and RA/TPA differentiated
cells show significantly lower levels of VMAT as compared to undifferentiated cells or RA treatment alone
(FIG. 3-C). As compared to UN cells the RA, TPA and
RA/TPA had significant but relatively small increases
in -actin and MAP-2 expression (FIG. 4-F, 4-G), indicating that increased levels of TH and DAT were not
simply due to increased neuronal growth induced by
TPA/TPA or RA/TPA. While protein levels for the D2
and D3 receptor were almost undetectable in UN cells,
the differentiation conditions significantly altered the
levels of expression (FIG. 3-D, 3-E). RA, TPA and
RA/TPA treatment elevated protein levels for the D2
receptor, and TPA/TPA differentiation and RA/TPA
differentiation increased the amount of D3 receptor by
3 and 6-fold, respectively (FIG. 3-E).
Kinetics of Uptake of [3H]DA and [3H]MPP+ in
RA/TPA and RA/RA Differentiated Cells
To determine if the high levels of DAT in the RA/TPA
treated cells were functionally similar to that in vivo,
we compared the kinetics of [3H]DA and [3H]MPP+
uptake in RA/TPA differentiated cells as compared
with that of mouse and rat synaptosomal preparations
of caudate-putamen containing DA terminals. In preliminary experiments, we optimized the assay conditions for specific [3H]DA and [3H]MPP+ uptake to DAT
in mouse, rat and SH-SY5Y synaptosomes. Maximal
uptake rates were determined for all groups (FIG. 4-A,
4-B, 4-C); the Vmax values for [3H]DA uptake for mouse
(36.69 15.4 pmol/mg protein) and rat (32.82 5.8
pmol/mg protein) were almost identical and similar to
that reported by Eshleman and associates (Eshleman et
585
FIGURE 1 Immunocytochemical detection of dopaminergic markers in RA/TPA differentiated SH-SY5Y Cells. Immunocytochemistry and
confocal 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 D2 receptor polyclonal antibody.
Panel E is RA/TPA cells incubated with rabbit anti-dopamine D3 receptor polyclonal antibody.
Panels A1-E1 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). Levels
of D2 (D) and D3 (E) receptor levels also are higher in RA/TPA differentiated cells.
586
FIGURE 2 Immunocytochemical detection of dopaminergic markers in RA differentiated SH-SY5Y cells. Immunocytochemical and
laser scanning microscopy were used as described in Materials and Methods. Panels A-E are differential interference contrast (DIC)
images of RA differentiated SH-SY5Y cells as described in Fig. 1. Panels A2-E2 are composite images of MAP-2 (A1-E1) with the corresponding DA-ergic marker (A2-E2).
al., 2001). The Km values were also similar to that

reported by Eshleman and associates (Eshleman et al.,
2001) and not significantly different in mouse and rat
caudate-putamen preparations. The Vmax value for
[3H]DA uptake in RA/TPA treated SH-SY5Y cells
(33.69 9.5 pmol/mg protein) was also very similar to
that derived from mouse and rat caudate-putamen
preparations but the Km was higher. Direct comparison

of Vmax and Km values in RA/TPA treated cells with
RA/RA treated cells (FIG. 4-B) showed that the Vmax
was significantly higher in the RA/TPA treated cells (ttest, P <0.001) as was the Km (t-test, P <0.001). While
the Vmax for [3H]MPP+ uptake in SH-SY5Y cells (FIG.
5-C) was slightly higher (42.47 9.5 pmol/mg protein)
587
than for [3H]DA uptake, they were not statistically different.
Figure 3
MPP+ and DA Neurotoxicity

It has been proposed that the relative levels of expression of DAT and VMAT critically regulate the sensitivity of DAergic neurons to MPP+ (Przedborski et al.,
2000) in vivo. To test further if the relative levels off
DAT and VMAT in the SH-SY5Y cells under varying
differentiation conditions altered their sensitivity to
MPP+ in RA as compared to RA/TPA differentiated
cells, the cytotoxicity of MPP+ was assayed over a fourday period at 24-h intervals, utilizing sister cultures in
the MTT assay that measures mitochondrial function
(FIG. 5-A, 5-B, 5-C). There were significant treatment
(cell type, P <0.05) and time ((P <0.05) differences. At
the lowest concentration of MPP+ the RA/TPA differentiated cells exhibited greater cytotoxicity than the
RA differentiated cells at all time points (FIG. 5-A). At
higher concentrations of MPP+ the selectivity was
reduced, so that at 0.3 mM MPP+ differences between
RA/TPA and RA differentiated cells existed only for 48
h (FIG. 5-B). At the highest concentration of MPP+ (3
mM) RA/TPA and RA differentiated cells showed significant differences in mitochondrial dysfunction in
response to MPP+ only at the 24 h test point (FIG. 5-C).
We also characterized cell death to DA in the RA and
RA/TPA differentiated SH-SY5Y cells. Varying concentrations of DA (0 to 500 M) were added to the
media and at 24 h intervals the cell viability was determined with LDH release (FIG. 5-D). There were not
significant treatment (cell type, P > 0.5) but there were
significant effects of concentration ((P <0.05). There
was significant cytotoxicity exhibited at concentrations
of 150 M and greater in both RA and RA/TPA differentiated cells, with no apparent differences in sensitivity between cell types (FIG. 5-D, 5-E).
To further characterize differences in DA and MPP+
induced cell death in RA/TPA differentiated cells,
FIGURE 3 Protein levels for TH, DAT and VMAT-2, D2 receptor, D3 receptor, b-actin and MAP-2 in UN, RA, TPA and RA/TPA differentiated SH-SY5Y cells. For panels A-G the western blot and resulting densitometric analysis are shown for different antibodies as
described in Materials and Methods. The results are representative of n =3 replications. Each lane of the 4%/10% SDS-PAGE gels was
loaded with 20 M of protein from the cells. A. The resultant PVDF membrane was incubated with rabbit anti-tyrosine hydroxylase affinity purified polyclonal antibody (Chemicon AB152) at a 1:1000 dilution overnight at 4C; a distinct band was present at ~59kD. B. The
membrane was incubated with rat anti-dopamine transporter monoclonal antibody (Chemicon MAB369) at a 1:1000 dilution overnight at
4C; a distinct band was present at ~80kD. C. The membrane was incubated with rabbit anti-vesicular monoamine transporter 2 affinity
purified polyclonal antibody (Chemicon AB 1598P) at a 1:1000 dilution overnight at 4C; a distinct band was present at ~50kD. D. The
resultant PVDF membrane was incubated with rabbit anti-dopamine D2 receptor polyclonal antibody (Chemicon AB1558) at a 1:1000 dilution overnight at 4C; a distinct band was present at ~50kD. E. The membrane was incubated with rabbit anti-dopamine D3 receptor affinity purified polyclonal antibody (Chemicon AB1785P) at a 1:1000 dilution overnight at 4C; a distinct band was present at ~50kD. F. The
resultant PVDF membrane was incubated with mouse anti-b-actin monoclonal antibody (Sigma A5441) at a 1:1000 dilution overnight at
4C; a distinct band was present at ~42kD. G. The membrane was incubated with mouse anti-MAP-2 monoclonal antibody (Chemicon
MAB3418) at a 1:1000 dilution overnight at 4C; a distinct band was present at ~300kD. Lane Analysis (densitometry) was performed using
AIS 6.0. *P <0.001 vs UN.
588
FIGURE 4 [3H]DA or [3H]MPP+ uptake in synaptosomes prepared

from 6 week old mouse, 10-week-old rat and SH-SY5Y neuroblastoma cells. Data shown are the mean S.E.M. from rat (n = 3) and
mouse (n = 6) striatum (A), or RA/RA as compared to RA/TPA differentiated SH-SY5Y cells (B), or RA/TPA differentiated SHSY5Y cells (C), each conducted in triplicate. Km and Vmax were
determined by nonlinear regression. The Vmax values from mouse
and rat were not significantly different. The Vmax and Km for
[3H]DA uptake were significantly different between RA/RA and
RA/TPA cells. The Vmax [3H]DA or [3H]MPP+ uptake in SH-SY5Y
cells showed no significant difference. Differences between groups
were tested for significance using One-way ANOVA followed by
Bonferonni's post-hoc analysis.
varying concentrations of DA (0 to 500 M) were

added to the media and at 24 h intervals the cell viability was determined in sister cultures utilizing cell
counts, LDH release and MTT assay (FIG. 6). There
were significant treatment (concentration, P <0.05) and
time effects ((P <0.05). There was consistent cell death
at concentrations of 150 M and greater when measured by cell counts and LDH release (FIG. 6-A, 6-B).
Mitochondrial dysfunction was also impacted by 150
M DA (FIG. 6-C), but not until after 48 h when significant cell death had already been detected by cell
counts and LDH release. Varying concentrations of DA
(0 to 500 M) were added to the media with and without RA/TPA differentiated SH-SY5Y cells and the formation of H2O2 in the media was measured at 3, 6, 12,
24, 48, 72 and 96 h time points (data not shown). At 24,
48, 72 and 96 h significant increases in extracellular
H2O2 was detected for concentrations of 150 M and
greater. The levels of H2O2 were dependent on the concentration of DA but were independent of time from 48
to 96 h. There was also no significant difference in the
levels of H2O2 generated from DA in the presence or
absence of cells, suggesting autooxidation was taking
place.
To determine if the ROS-generating neurotoxic
effects of DA and neurotoxic effects of MPP+ were
additive, we examined the impact of a subthreshold
concentration of DA (100 M), the LD50 concentration
of MPP+ (100 M) and the combination of DA and
MPP+ for LDH release (FIG. 7-A) and mitochondrial
dysfunction (FIG. 7-B) in RA/TPA differentiated SHSY5Y cells. There were significant treatment (drug, P
<0.05) and time effects ((P <0.05). As shown in fig. 7A, LDH release was significantly increased by MPP+ at
72 and 96 h post treatment, and it was augmented by
the addition of a subthreshold concentration of DA that
was not cytotoxic on its own. In contrast, mitochondrial dysfunction was induced by MPP+ but not by DA,
and the effects of MPP+ and DA were not additive. The
intracellular levels of ROS was determined at 6, 24, 48
and 72 h after the addition of varying concentrations off
MPP+ (FIG. 7-C) or DA (FIG. 7-D) to sister cultures.
There were significant treatment (concentration, P
<0.05) and time effects ((P <0.05) for addition of DA to
the cultures. Concentrations below or above the LD50
concentration of MPP+ did not affect intracellular levels of ROS. In contrast, concentrations of DA at 50 M
and greater did significantly increase intracellular ROS
at 48 and 72 h time points.
589
90
80
70
60
50
40
30
20
10
0
RA
R/T
*&
*q
#
24
48
72
96
C ell death with DA at 48 hrs
4000
3500
Percent LDH Release
Percent Cell Death
A 100MPP+ Toxicity at 0.03 mM
RA/RA
3000
RA/TPA
2000
1500
1000
500
h ours
100
90
80
70
60
50
40
30
20
10
0
*q
*#
24
48
72
96
h ours
Percent Cell Death
M PP+ Toxicity at 3 mM
100
90
80
70
60
50
40
30
20
10
0
10 50 100 150 250 500
Concentration DA [uM]
M PP+ Toxicity at 0.3 mM
Percent LDH Release
Percent Cell Death
2500
C ell Death with DA at 72 hrs

4500
y
4000
3500
RA/RA
3000
RA/TPA
2500
2000
1500
1000
500
0
0
10 50 100 150 250 500
Concentration DA [uM]
24
48
72
96
h ours
FIGURE 5 Neurotoxic effects of MPP+ (A-C) and DA (D, E) in RA and RA/TPA differentiated SH-SY5Y cells. SH-SY5Y cells were
treated with different concentrations of MPP+ (0, 0.03, 0.3 and 3.0 mM) or DA (0 to 500 M) 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 LDH
release (D, E). RA cells were resistant to MPP+ at all but the highest concentration of MPP+. In contrast, the RA/TPA differentiated cells
exhibited pronounced effects at the lowest concentration of MPP+ by 24 h post-MPP+ and maintaining the sensitivity at 72 and 96 h (A). At
the higher concentrations the differences in sensitivity between differentiation conditions existed at 24 and 48 h but not 72 and 96 h postMPTP. The cytotoxic effects of DA existed at concentrations of 150 M and greater but did not differ in sensitivity between RA and RA/TPA
differentiated cells. The values are the mean standard deviation (n= 3), two-way ANOVA showed significant treatment (cell type, P <0.05)
and time ((P <0.05) differences. Significance: *, P <0.001 for RA vs RA/TPA; #, P <0.001 for RA/TPA at 24 h vs 0 time point; q, P <0.001
for RA/TPA at 48 h vs 24 h time point; &, P <0.001 for RA/TPA at 72 h vs 48 h time point; Y, P <0.001 for RA and RA/TPTA vs 0 concentration DA.
590
FIGURE 6 Effects
f
of DA on cell death (A), LDH release (B) and
mitochondrial dysfunction (C) are shown. Varying concentrations
of DA were added to the media containing RA/TPA differentiated
SH-SY5Y cells and cell viability measured at 24 h intervals. The
results are shown as the mean S.D. (in triplicate) for different
concentrations of DA as a function of time from treatment at 24 h
intervals. Note that at 150 M DA significant toxicity is evident by
24 h as measured by cell death (A) and LDH release (B) but not
inhibition of MTT until 72 h post treatment. *, denotes significant
difference from no DA treatment (0 conc) at P <0.01.
Effect of Transporter Inhibiitors on Neurotoxicity

Induced by MPP+ and DA
To determine if MPP+ toxicity required intracellular
accumulation via DAT, as it does in vivo (Gainetdinov
et al., 1997), RA/RA and RA/TPA differentiated cells
were treated concurrently with the selective DAT
blocker GBR 12909 (Matecka et al., 1996) in the presence of different concentrations of MPP+. There were
significant treatment (cell type, P <0.05) and concentration effects ((P <0.05). As noted previously, RA/TPA
differentiated cells exhibited greater sensitivity than

RA/RA cells at 48 h post MPP+ (FIG. 8, P <0.001).
Concurrent treatment of SH-SY5Y cells with the DAT
blocker GBR 12909 (conc. 0.1 and 1.0 M) to the
RA/TPA differentiated cells demonstrated that blockade of DAT reduced neurotoxicity to MPP+ as measured by the MTT assay by up to 86% (FIG. 8). Thus,
consistent with our evidence that MPP+ uptake demonstrates kinetics similar to DA uptake via DAT, as the
neurotoxicity of MPP+ in the RA/TPA differentiated
cells is almost completely dependent on intracellular
accumulation via the DA transporter. The neurotoxic
effect of MPP+ was not significantly antagonized by
GBR 12909 in the RA cells, and the degree of inhibition was significantly less than in the RA/TPA cells ((P
<0.001). This is consistent with the lower expression off
DAT in these cells. It would also suggest that some off
the intracellular accumulation in RA differentiated
cells was via non-DAT mechanisms (Martel et al.,
2000).
To determine if the neurotoxicity of RA/TPA differentiated SH-SY5Y cells by DA required intracellular
transport as it does for MPP+, cell viability was measured after addition of 200 M DA and 10 M of the
DAT inhibitor GBR 12909, the NE transport inhibitor
nisoxetine and the 5-HT transport inhibitor 6-nitroquipazine or combinations of the transport inhibitors.
There were no significant treatment (drug, P >0.5) but
were significant effects of time ((P <0.05). DA produced significant reductions in cell number (FIG. 9-A)
and depletion of extracellular GSH (FIG. 9-B) by 24 h
post treatment. Extracellular levels of H2O2 did not
increase until after 24 h (FIG. 9-C). None of the transport inhibitors alone or in combination affected the
degree or temporal pattern of cell death, extracellular
GSH depletion, or formation of H2O2 by DA.
Pretreatment with Pramipexole is Neuroprotective
Against MPP+ in RA/TPA Cells
Pretreatment with the D3 preferring agonist pramipexole for 72 h significantly reduced MPP+ neurotoxicity
to the UN SH-SY5Y cells, but appeared to be non-DA
receptor mediated (Kitamura et al., 1998). To test that
neuroprotective effects of pramipexole were different
in RA (Low D2/D3 receptor) and RA/TPA differentiated (high D2/D3 receptor) cells we pretreated the cells
with pramipexole for 72 h prior to treatment with
MPP+. There were significant treatment (cell type, P
<0.05) and concentration effects ((P <0.05). As previously described, RA/TPA treated cells were more sensitive to the effects of each concentration of MPP+ (P
(
<0.001) using either a measure of membrane disruption
591
FIGURE 7 The neurotoxic effects of DA and MPP+ (A, B) and intracellular generation of ROS (C, D) are shown. The additive effects for
LDH release (A) but not for mitochondrial dysfunction (B) of subthreshold concentration of DA plus the LD50 concentration of MPP+ are
shown. The results are depicted as the mean standard deviation (in triplicate) for MPP+ alone (100 M), DA alone (100 M) and MPP+
with DA as a function of time from treatment at 24 h intervals. In RA/TPA differentiated SH-SY5Y cells loaded with CM-H2DCFDA the
level of ROS was not increased with any concentration of MPP+ (C) but was increased by DA (D) at concentrations of 50 M and greater.
Two-way ANOVA showed significant treatment (drug, P <0.05) and time ((P <0.05) differences. For bar graph A, # denotes significant difference from DA treatment at P <0.01. * denotes significant difference from MPP+ and DA treatment at P <0.01. For bar graph B, * denotes
significant difference from MPP+ and MPP+ with DA treatment at P <0.01. For bar graph D, * denotes significant difference from no DA (0
concentration) treatment at P <0.01.
(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 low
concentration of MPP+ was reduced by more than 50%
and was significant even at the highest concentration of
MPP+. In contrast, RA treated cells exhibited concentration-dependent cell death that was not attenuated by
pretreatment with PPX.
To determine if pretreatment was necessary for the
neuroprotective effects of pramipexole RA and
RA/TPA differentiated cells were treated concurrently
or 72 h prior to MPP+. There were significant treatment
(6 groups, P <0.05) and concentration effects ((P
<0.05). Pretreatment of SH-SY5Y cells with pramipexole (1.0 mM) for 72 h (FIG. 10-C) before addition of
differing concentrations of MPP+ to the RA/TPA differentiated cells reduced neurotoxicity as measured by the
MTT assay by around 55% (P
( <0.001). Concurrent
treatment with pramipexole did not afford significant
protection against MPP+ in the RA/TPA differentiated
cells at the lowest concentration but provided modest

(~20%) but significant attenuation at the highest concentration (1.0 mM) of MPP+. Neither concurrent nor
pretreatment with pramipexole provided protection
against MPP+ in the RA differentiated cells.
To determine if the neuroprotective effects off
pramipexole were DA receptor mediated RA/TPA differentiated cells we tested if the neuroprotective effects
of pramipexole were concentration-dependent and
could be antagonized with D2-like or D1-like receptor
blockers. RA/TPA differentiated cells were administered a range of concentrations of pramipexole in the
presence of a single concentration of MPP+ (0.1 mM).
As shown in FIG. 11-A, pramipexole pretreatment
demonstrated a concentration-response dependent neuroprotection at 48 h post MPP+ administration with an
EC50 of 41 M. RA/TPA treated SH-SY5Y cells were
then administered with a single concentration off
pramipexole (1 mM), approximately 2.5 times the
EC50, 3 days prior to initiation of MPP+ treatment in the
592
FIGURE 8 GBR 12909 blocks neurotoxic effects of MPP+ in

RA/TPA differentiated SH-SY5Y cells. RA and RA/TPA differentiated SH-SY5Y cells were treated with different concentrations of
MPP+ (0, 0.03, 0.3 and 3.0 mM) in the presence and absence of different concentrations of the dopamine transporter blocker
GBR12909 (0, 0.1, 1.0 M) and the neurotoxic effects of MPP+
monitored at 48 h by MTT assay. Note that the neurotoxic effects
of MPP+ were greater in the RA/TPA as compared to the RA differentiated cells and GBR 12909 completely antagonized the
effects of MPP+ in RA/TPA differentiation conditions. The values
are the mean standard deviation (n=3), two-way ANOVA showed
significant treatment (cell type, P <0.05) and concentration effects
( <0.05). Significance: *, P <0.001 vs 0 mM MPP+; q, P <0.001
(P
for RA vs RA/TPA ; #, P <0.001 for 0 concentration GB12909 vs
0.1, 1.0 M concentrations.
presence or absence of a D2/D3/D4 antagonist (spiperone), D3 preferring antagonist (U99194A), or the D1

receptor antagonist SCH 23390 (FIG. 11-B). There
were significant treatment (drug, P <0.05) and time
effects ((P <0.05). Pretreatment with pramipexole was
effective in reducing the neurotoxic effects of MPP+ in
RA/TPA treated cells, and that neuroprotection was
completely blocked by a high concentration of spiperone (100 M, P <0.001) and a low concentration of the
D3 preferring antagonist U99194A (10 M, P <0.001).
SCH 23390 at a high concentration (100 M) was ineffective in blocking the effects of pramipexole.
DA induced toxicity was also attenuated by
pramipexole but concurrent treatment was as effective
as pretreatment (data not shown). Treatment with either
100 M or 1 mM pramipexole produced a similar
reduction in cell death (FIG. 11-C) and this effect of
pramipexole was not inhibited by a high concentration
of spiperone or SCH 23390.
Figure 9
FIGURE 9 The cell death, extracellular GSH depletion and formation of H2O2 caused by DA are not attenuated by the transporter
inhibitors for NET, SERT and DAT. The results are shown as the
mean standard error (in triplicate) for DA alone (200 M), or
with nisoxetine (NET antagonist), 6-nitroquipzine (SERT antagonist), GBR 12909 (DAT antagonist), or combinations of the transport inhibitors as a function of time from treatment at 6, 12, 24, 48,
72 and 96 h. At no time point was any of the drugs able to reduce
the cytotoxicity, extracellular GSH depletion or formation of H2O2
by DA. Abbreviations: GBR/Nis, GBR 12909 with Nisoxetine;
GBR/6-Nit, GBR 12909 with 6-Nitroquipazine; Nis/6-NIT,
Nisoxetine with 6-Nitroquipazine; GBR/Nis/6-Nit, GBR 12909
with Nisoxetine and 6-Nitroquipazine
593
DISCUSSION
SK-N-SH cell line, established in the early 1970s, was
derived from malignant tumors of immature neurons
(Ross et al., 1983) and has been shown to exhibit properties of stem cells, consisting of a heterogeneous population of cell types that interconvert. While SK-N-SH
cells express enzymes for DA and NE synthesis
(Oyarce and Fleming, 1991), they have been reported
to secrete more DA than NE in vitro (Richards and
Sadee, 1986). These cells have since been cloned to
create the line known as SH-SY5Y, which maintains
stem cell characteristics (Ross et al., 1983) and is capable of proliferating in culture for long periods without
contamination (Biedler et al., 1978; Ross et al., 1983;
Willets et al., 1995). These cells have been utilized to
study mechanisms of apoptosis by MPP+ and neuroprotection by DA agonists (Fang et al., 1995; Sheehan et
al., 1997; Song et al., 1997; Kitamura et al., 1998).
Apoptosis produced by MPP+ is correlated with formation of ROS in the mitochondria, disrupted electron
transport, collapse of the mitochondrial potential,
release of cytochrome c from the organellar to the
cytosolic fraction, followed by DNA laddering (Itano
and Nomura, 1995; Fall and Bennett, Jr. 1999; Veech et
al., 2000; Kitamura et al., 1998; Kakimura et al.,
2001). Concurrent treatment with pramipexole
(Cassarino et al., 1998) or pretreatment with DA agonists bromocriptine, talipexole and pramipexole for 4
days significantly reduced MPP+ neurotoxicity to the
neuroblastoma SH-SY5Y cell line (Kitamura et al.,
1998). A high concentration of pramipexole reduced
the levels of ROS caused by MPP+ and stabilized the
mitochondrial transition pore (Cassarino et al., 1998).
Pretreatment for 4 days with pramipexole or talipexole
was shown to increase levels of the anti-apoptotic protein Bcl-2 and Bcl-XL, prevent the translocation of
cytochrome c from the organellar to the cytosolic fracFIGURE 10 Pramipexole pretreatment antagonizes the effects of MPP+ in RA/TPA but not RA differentiated SH-SY5Y cells. RA and
RA/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 differentiated 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 <0.05) and concentration
( <0.05) effects. Significance: *, P <0.001 vs 0 mM MPP+; q, P <0.001 for RA vs RA/TPA. Abbreviations: R/T, RA and TPA differentiat(P
ed cells given vehicle; PPX, RA and TPA differentiated cells given pramipexole. For C, note that significant neurotoxicity occurred at 0.3
and 1.0 mM MPP+ that was antagonized by pretreatment with pramipexole. At the highest does of MPP+, cotreatment with pramipexole produced small but significant reduction of the neurotoxic effects of MPP+. The values are the mean standard deviation (n=3). Significance:
*, P <0.001 vs 0 mM MPP+; F, P <0.001 for No PPX vs CoPPX. Abbreviation: RA-Veh, RA differentiated cells with vehicle treatment;
RA-CoPPX, RA differentiated cells with concurrent pramipexole and MPP+ treatment; RA-PrePPX, RA differentiated cells with 3 day pretreatment with pramipexole prior to MPP+ treatment; R/T-Veh, RA and TPA differentiated cells with vehicle treatment; R/T -CoPPX, RA
and TPA differentiated cells with concurrent pramipexole and MPP+ treatment; R/T -PrePPX, RA and TPA differentiated cells with 3 day
pretreatment with pramipexole prior to MPP+ treatment.
594
tion produced by MPP+, inhibit cytochrome c activation of caspase-9, and prevent DNA laddering produced by MPP+ (Kitamura et al., 1998; Kakimura et al.,
2001). D2-selective and non-selective antagonists did
not, however, inhibit the neuroprotective effects of talipexole or pramipexole against MPP+-induced apoptosis of SH-SY5Y cells (Kitamura et al., 1998), and free
radical scavenging properties of these compounds have
been favored as the mechanism of neuroprotection.
This might invalidate the use of SH-SY5Y cells to
study the neuroprotective effects of DA agonists, since
DA receptor effects in vivo do utilize DA receptor
mediated effects.
There are problems with use of the undifferentiated
SH-SY5Y cells as a model for dopaminergic neurons
as there is not high expression of DA synthetic
enzymes, toxicity by MPP+ does not require DAT, and
ongoing mitosis can dramatically affect the response to
drugs and toxins. It had been previously reported that
RA differentiation of SH-SY5Y cells followed by treatment with the phorbol ester TPA significantly elevates
production of DA synthetic enzymes (Pennypacker et
al., 1989), D2 receptors (Farooqui 1994) and its G-proteins (Ammer and Schulz 1994). We confirmed this by
identifying that SH-SY5Y cells were immunoreactive
to TH and DAT antibodies, and that membranes prepared from the SH-SY5Y cells were also immunopositive using western blotting. Terminal differentiation
with RA followed by TPA treatment significantly
increased the amount of TH and DAT as compared to
UN, or RA differentiated cells. In contrast, combined
treatment with RA and TPA actually reduced levels off
VMAT. To test the appropriateness of the RA/TPA differentiated cells as a dopaminergic model cell line we
further determined that the RA/TPA cells were sensitive to concentrations of MPP+ lower than that required
for undifferentiated SH-SY5Y cells (above 1 mM)
(Fall and Bennett, Jr., 1999)), and that treatment off
cells with the DAT antagonist GBR 12909 resulted in
an almost complete blockade of the toxicity 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 MTT
assay. The values are expressed as percentage of the MTT inhibition in cells incubated with MPP+ only and are the mean standard deviation (n=3). Significance protection from MPP+ at * P <0.05. The EC50 for protection by PPX was 41 M (determined by nonlinear regression, sigmoidal concentration response using Graphpad Prism 3.0). B. RA/TPA differentiated SH-SY5Y cells were treated with a single concentration of MPP+ (0.3 mM) or media alone 3 days after exposure to pramipexole (PPX, 1.0 mM) or vehicle (VEH) in the presence or
absence of spiperone (10 M), U99194A (1 M) or SCH 23390 (10 M). The neurotoxic effects of MPP+ monitored at 48 h by MTT assay.
The values are the mean standard deviation (n=3). Significance: *, P <0.001 for Pre PPX vs spiperone and U99194A but not SCH 23390.
C. RA/TPA differentiated SH-SY5Y cells were treated with a single concentration of DA (150 M) or media alone 3 days after exposure to
pramipexole (PPX, 100 M and 1.0 mM) or vehicle (VEH) in the presence or absence of spiperone (10 M), or SCH 23390 (10 M). The
neurotoxic effects of DA monitored at 48 h by cell counts. The values are the mean standard deviation (n=3). Significance: #, P <0.001
for DA alone vs DA plus PPX (100 M or 1 mM); *, P <0.001 for spiperone and SCH 23390 vs DA alone.
This is in contrast to undifferentiated SH-SY5Y cells

(Fang et al., 1995; Willets et al., 1995), but similar to
what occurs in primary cultures of mesencephalic
dopaminergic neurons (Bilsland et al., 2002).
Furthermore, we found that RA/TPA differentiated SHSY5Y cells exhibited specific DAT mediated DA and
MPP+ uptake with very similar kinetics to mouse and
rat striatal synaptosomes containing DA terminals. Our
results with mouse and rat striatal synaptosomes are
similar to that reported by Eshleman and associates
(Eshleman et al., 2001), and the comparability between
the RA/TPA differentiated SH-SY5Y cells and mouse
and rat striatal synaptosomes is indicative that these
cells exhibit high native expression of a functional
form of DAT. The higher Km in the cells as compared
to the mouse and rat striatal synaptosome likely is due
to the absence of catechol-O-methyltranferease in the
cells, which is known to alter the kinetics of DA transport (Eshleman et al., 1997). In contrast to the RA/TPA
differentiated cells, RA/RA differentiated cells exhibited significantly less DA uptake, were less sensitive to
MPP+-induced toxicity, and the MPP+ toxicity was not
inhibited by a DAT blocker. This is similar to undifferentiated SH-SY5Y cells in which inhibition of catecholamine uptake does not block MPP+-induced neurotoxicity, indicative of why such high concentrations of
MPP+ are required to induce apoptosis (Fang et al.,
1995; Willets et al., 1995). Our data support the proposal that the ratio of DAT to VMAT can modulate the
sensitivity of SH-SY5Y cells to MPP+, as it does in vivo
(Gainetdinov et al., 1997). Thus, RA/TPA differentiated SH-SY5Y cells exhibit characteristics consistent
with cultured DA neurons, but represent a more feasible model to explore the mechanisms of cell death produced by MPP+.
While DA agonist mechanisms providing neuroprotection are varied and probably include their free-radical scavenging properties (Cassarino et al., 1998;
Kitamura et al., 1998; Ling et al., 1998; Grnblatt et
al., 1999), it would be important to demonstrate DA
receptor mediated effects in cell culture. Our results
suggest that the robust expression of D2 and, particularly, D3 receptors in the RA/TPA differentiated cells is
necessary for DA receptor-mediated protection by
pramipexole. Consistent with what has been observed
in undifferentiated SH-SY5Y cells we determined that
pretreatment of SH-SY5Y cells with the D3 preferring
agonist pramipexole for three days before addition of
MPP+ to the RA/TPA differentiated cells reduced MPP+
neurotoxicity as measured by the MTT assay in the
RA/TPA differentiated cells. While a similar degree of
protection was measured by LDH release, the degree of
595
cell loss measured with this protocol is less than that

with MTT at the same time points. Cells with disturbed
mitochondrial function, as determined by the MTT
assay, may not be exhibiting cell lysis or membrane
disruption, as measured by the LDH release assay, until
later time points. Importantly, concurrent treatment
with pramipexole did not afford protection to the neurotoxic effects of MPP+ in the RA/TPA treated cells and
neither pretreatment nor concurrent treatment was
effective in RA differentiated cells. The RA differentiated cells with low expression of D2 and, particularly,
D3 receptors exhibited negligible protection by
pramipexole. We demonstrated concentration-dependent effects for neuroprotection by pramipexole, consistent with receptor mediated effects, and that a D2/D3/D4
or D3 preferring receptor blocker potently antagonized
the neuroprotective effects of pramipexole. In contrast,
the D1 receptor antagonist SCH 23390 was ineffective.
Thus, it is unlikely that the neuroprotective effects are
solely dependent on the antioxidant capabilities off
pramipexole and support evidence by Carvey and Ling
(1997), that at least some of the neuroprotective effects
of pramipexole are D3 receptor mediated. The coexpression of D2 and D3 receptors in the RA/TPA differentiated cells is phenotypically similar what exists in
the brain, in which DAergic neurons express D2 and D3
receptors (Le Moine and Bloch 1991; Diaz et al., 2000)
and both of which are functional (Gobert et al., 1995;
Koeltzow et al., 1998; L'hirondel et al., 1998;
Dickinson et al., 1999; Jung et al., 1999; Zapata et al.,
2001; Zapata and Shippenberg, 2002). Hence, terminally differentiated SH-SY5Y cells make a good model
for what occurs in vivo, and the role of DA receptormediated neuroprotection against MPP+ can be more
clearly defined.
Our data also indicate that DA-induced cell death and
that by MPP+ in the RA/TPA differentiated cells occurs
via different mechanisms. It has been previously shown
in a variety of cell lines that excess DA added to the
media will produce cell death associated with depletion
of GSH and an intracellular rise in ROS (Masserano et
al., 1996; Lai and Yu 1997; Jones et al., 2000; Stokes
et al., 2000). We show here that excess DA added to the
media increases the formation of H2O2 and reduction off
GSH, and a concomitant increase in intracellular ROS
and reduction of GSH. In contrast, MPP+ does not produce an intracellular rise in ROS, nor was it additive to
the ROS effects of DA. Interestingly, neither the cell
death nor rise in intracellular ROS is affected by blockade of DA, NE, 5-HT or a combination of blockers off
uptake. This is inconsistent with some studies
596
(Simantov et al., 1996; Iida et al., 1999; Jones et al.,

2000; Le et al., 2000) but is consistent with other studies (Iida et al., 1999; Koshimura et al., 2000; Le et al.,
2000; Storch et al., 2000b). It may well be that DA cell
death is associated with activation of JNK and p38
(Gmez-Santos et al., 2003) and separate from caspase
dependent cell death induced by MPP+ (Itano and
Nomura 1995; Kitamura et al., 1998; Fall and Bennett,
Jr., 1999; Veech et al., 2000; Gmez et al., 2001;
Kakimura et al., 2001). Our results also confirm other
studies that a variety of D2/D3 agonists are effective in
attenuating the cytotoxic effects of DA that is associated with prevention of the depletion of GSH or an
increase in levels of GSH (Iida et al., 1999; Le et al.,
2000). Since the effects of the D2/D3 agonists were not
antagonized by spiperone or SCH 23390, it would suggest that the actions of the D2/D3 agonists are not
dependent on DA receptor mediation. However, as
spiperone and SCH 23390 attenuated the cytotoxic
effects of DA by themselves, it remains an open question as to whether there is any DA receptor mediation
in the protective effects of the D2/D3 agonists. The
RA/TPA differentiated cells appear to be an excellent
model system to study the receptor dependence of protection by DA agonists against MPP+ and DA induced
cyctoxicity.
Acknowledgments
Funded by Federal Grant NS40669, Arizona
Alzheimer's Disease Research Center contract
4001(Arizona Parkinson's Disease Center), and
Pharmacia Corporation (Kalamzaoo, MI). This paper is
submitted in partial fulfillment of the requirements for
a doctoral thesis at Arizona State University for SP. The
assistance of Dr. Mateo Paz Soldn in immunocytochemistry and confocal microscopy is gratefully
acknowledged.
References
Ammer H and R Schulz (1994) Retinoic acid-induced differentiation of human neuroblastoma SH-SY5Y cells is associated with
changes in the abundance of G proteins. J. Neurochem. 62, 13101318.
Anderson DW, T Neavin, JA Smith and JS Schneider (2001)
Neuroprotective effects of pramipexole in young and aged
MPTP-treated mice. Brain Res. 905, 44-53.
Biedler JL, S Roffler-Tarlov, M Schachner and LS Freedman
(1978) Multiple neurotransmitter synthesis by human neuroblastoma cell lines and clones. Cancer Res. 38, 3751-3757.
Bilsland J, S Roy, S Xanthoudakis, DW Nicholson, Y Han, E
Grimm, F Hefti and SJ Harper (2002) Caspase inhibitors attenuate 1-methyl-4-phenylpyridinium toxicity in primary cultures of
mesencephalic dopaminergic neurons. J. Neurosci. 22, 26372649.

Blum D, S Torch, N Lambeng, M Nissou, AL Benabid, R Sadoul
and JM Verna (2001) Molecular pathways involved in the neurotoxicity of 6-OHDA, dopamine and MPTP: contribution to the
apoptotic theory in Parkinson's disease. Prog. Neurobiol. 65,
135-172.
Burns RS, CC Chiueh, SP Markey, MH Ebert, DM Jacobowitz and
IJ Kopin (1983) A primate model of parkinsonism: selective
destruction of dopaminergic neurons in the pars compacta of the
substantia nigra by N
N-methyl-4- phenyl-1,2,3,6-tetrahydropyridine. Proc. Natl. Acad. Sci. USA 80, 4546-4550.
Carvey PM and ZD Ling (1997) The case for neuroprotection with
dopamine agonists. Clin. Neuropharmacol. 20[Suppl.], S8-S21.
Carvey PM, S Pieri and ZD Ling (1997) Attenuation of levodopainduced toxicity in mesencephalic cultures by pramipexole. J.
Neural Transm. 104, 209-228.
Carvey PM, SO McGuire and ZD Ling (2001) Neuroprotective
effects of D3 dopamine receptor agonists. Parkinsonism Relat.
Disord. 7, 213-223.
Cassarino DS and JP Bennett Jr (1999) An evaluation of the role of
mitochondria in neurodegenerative diseases: mitochondrial
mutations and oxidative pathology, protective nuclear responses,
and cell death in neurodegeneration. Brain Res. Brain Res. Rev.
29, 1-25.
Cassarino DS, CP Fall, TS Smith and JP Bennett Jr (1998)
Pramipexole reduces reactive oxygen species production in vivo
and in vitro and inhibits the mitochondrial permeability transition
produced by the parkinsonian neurotoxin methylpyridinium ion.
J. Neurochem. 71, 295-301.
Choi WS, Y Yoon, TH Oh, EJ Choi, KL O'Malley and YJ Oh (1999)
Two distinct mechanisms are involved in 6-hydroxydopamineand MPP+-induced dopaminergic neuronal cell death: role of caspases, ROS and JNK. J. Neurosci. Res. 57, 86-94.
Chun HS, GE Gibson, LA DeGiorgio, H Zhang, VJ Kidd and JH
Son (2001) Dopaminergic cell death induced by MPP+, oxidant
and specific neurotoxicants shares the common molecular mechanism. J. Neurochem. 76, 1010-1021.
Cohen G and P Werner (1994) Free radicals, oxidative stress and
neurodegeneration, in Neurodegenerative Disorders (Calne DB,
Ed.) (WB Saunders, Philadelphia, PA), pp 139-162.
Diaz J, C Pilon, B Le Foll, C Gros, A Triller, JC Schwartz and P
Sokoloff (2000) Dopamine D3 receptors expressed by all mesencephalic dopamine neurons. J. Neurosci. 20, 8677-8684.
Dickinson SD, J Sabeti, GA Larson, K Giardina, M Rubinstein, MA
Kelly, DK Grandy, MJ Low, GA Gerhardt and NR Zahniser
(1999) Dopamine D2 receptor-deficient mice exhibit decreased
dopamine transporter function but no changes in dopamine
release in dorsal striatum. J. Neurochem. 72, 148-156.
Eshleman AJ, E Stewart, AK Evenson, JN Mason, RD Blakely, A
Janowsky and KA Neve (1997) Metabolism of catecholamines
by catechol-O-methyltransferase in cells expressing recombinant
catecholamine transporters. J. Neurochem. 69, 1459-1466.
Eshleman AJ, K Wolfrum, DC Mash, K Christensen and A
Janowsky (2001) Drug interactions with the dopamine transporter in cryopreserved human caudate. J. Pharm. Exp. Ther.
296, 442-449.
Fall CP and JP Bennett Jr (1999) Characterization and time course
of MPP+ -induced apoptosis in human SH- SY5Y neuroblastoma
cells. J. Neurosci. Res. 55, 620-628.
Fang J, D Zuo and PH Yu (1995) Comparison of cytotoxicity of a
quaternary pyridinium metabolite of haloperidol (HP+) with neurotoxin N
N-methyl-4-phenylpyridinium (MPP+) towards cultured

dopaminergic neuroblastoma cells. Psychopharmacology (Berl.)
121, 373-378.
Farooqui SM (1994) Induction of adenylate cyclase sensitive
dopamine D2 -receptors in retinoic acid induced differentiated
human neuroblastoma SHSY-5Y cells. Life Sci. 55, 1887-1893.
Gainetdinov RR, F Fumagalli, SR Jones and MG Caron (1997)
Dopamine transporter is required for in vivo MPTP neurotoxicity: evidence from mice lacking the transporter. J. Neurochem. 69,
1322-1325.
Gobert A, JM Rivet, V Audinot, L Cistarelli, M Spedding, J Vian,
JL Peglion and MJ Millan (1995) Functional correlates of
dopamine D3 receptor activation in the rat in vivo and their modulation by the selective antagonist, (+)-S 14297: II. Both D2 and
"silent" D3 autoreceptors control synthesis and release in
mesolimbic, mesocortical and nigrostriatal pathways. J. Phar.
Exp. Ther. 275, 899-913.
Gmez C, J Reiriz, M Pique, J Gil, I Ferrer and S Ambrosio (2001)
Low concentrations of 1-methyl-4-phenylpyridinium ion induce
caspase- mediated apoptosis in human SH-SY5Y neuroblastoma
Gmez-Santos C, I Ferrer, AF Santidrian, M Barrachina, J Gil and
S Ambrosio (2003) Dopamine induces autophagic cell death and
alpha-synuclein increase in human neuroblastoma SH-SY5Y
Grnblatt E, S Mandel, T Berkuzki and MB Youdim (1999)
Apomorphine protects against MPTP-induced neurotoxicity in
mice. Mov. Disord. 14, 612-618.
Hirsch EC, S Hunot and A Hartmann (2000) Mechanism of cell
death in experimental models of Parkinson's disease. Funct.
Neurol. 15, 229-237.
Hornykiewicz O (1998) Biochemical aspects of Parkinson's disease. Neurology 51, S2-S9.
Iida M, I Miyazaki, K Tanaka, H Kabuto, E Iwata-Ichikawa and N
Ogawa (1999) Dopamine D2 receptor-mediated antioxidant and
neuroprotective effects of ropinirole, a dopamine agonist. Brain
Res. 838, 51-59.
Itano Y and Y Nomura (1995) 1-methyl-4-phenyl-pyridinium ion
(MPP+) causes DNA fragmentation and increases the Bcl-2
expression in human neuroblastoma, SH-SY5Y cells, through
different mechanisms. Brain Res. 704, 240-245.
Jones DC, PG Gunasekar, JL Borowitz and GE Isom (2000)
Dopamine-induced apoptosis is mediated by oxidative stress and
is enhanced by cyanide in differentiated PC12 cells. J.
Neurochem. 74, 2296-2304.
Jung M-Y, BV Skryabin, M Arai, S Abbondanzo, D Fu, J Brosius,
NK Robakis, HG Polites, JE Pintar and C Schmauss (1999)
Potentiation of the D2 mutant motor phenotype in mice lacking
dopamine D2 and D3 receptors. Neuroscience 91, 911-924.
Kakimura J, Y Kitamura, K Takata, Y Kohno, Y Nomura and T
Taniguchi (2001) Release and aggregation of cytochrome c and
alpha-synuclein are inhibited by the antiparkinsonian drugs, talipexole and pramipexole. Eur. J. Pharmacol. 417, 59-67.
Kirkland RA, JA Windelborn, JM Kasprzak and JL Franklin (2002)
A Bax-induced pro-oxidant state is critical for cytochrome c
release during programmed neuronal death. J. Neurosci. 22,
6480-6490.
Kitamura Y, Y Kohno, M Nakazawa and Y Nomura (1997)
Inhibitory effects of talipexole and pramipexole on MPTPinduced dopamine reduction in the striatum of C57BL/6N mice.
Jpn. J. Pharmacol. 74, 51-57.
Kitamura Y, T Kosaka, JI Kakimura, Y Matsuoka, Y Kohno, Y
Nomura and T Taniguchi (1998) Protective effects of the
antiparkinsonian drugs talipexole and pramipexole against 1-
597
methyl-4-phenylpyridinium-induced apoptotic death in human

neuroblastoma SH-SY5Y cells. Mol. Pharmacol. 54, 1046-1054.
Koeltzow TE, M Xu, DC Cooper, XT Hu, S Tonegawa, ME Wolf
and FJ White (1998) Alterations in dopamine release but not
dopamine autoreceptor function in dopamine D3 receptor mutant
mice. J. Neurosci. 18, 2231-2238.
Koshimura K, J Tanaka, Y Murakami and Y Kato (2000) Effects of
dopamine and L-DOPA on survival of PC12 cells. J. Neurosci.
Res. 62, 112-119.
L'hirondel M, A Cheramy, G Godeheu, F Artaud, A Saiardi, E
Borrelli and J Glowinski (1998) Lack of autoreceptor-mediated
inhibitory control of dopamine release in striatal synaptosomes
of D2 receptor-deficient mice. Brain Res. 792, 253-262.
Lai CT and PH Yu (1997) Dopamine- and L-beta-3,4-dihydroxyphenylalanine hydrochloride (L-Dopa)- induced cytotoxicity
towards catecholaminergic neuroblastoma SH-SY5Y cells.
Effects of oxidative stress and antioxidative factors. Biochem.
Pharmacol. 53, 363-372.
Langston JW, P Ballard, JW Tetrud and I Irwin (1983) Chronic
Parkinsonism in humans due to a product of meperidine-analog
synthesis. Science 219, 979-980.
Langston JW, LS Forno, J Tetrud, AG Reeves, JA Kaplan and D
Karluk (1999) Evidence of active nerve cell degeneration in the
substantia nigra of humans years after 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine exposure. Ann. Neurol. 46, 598-605.
Le WD, J Jankovic, W Xie and SH Appel (2000) Antioxidant property of pramipexole independent of dopamine receptor activation
in neuroprotection. J. Neural Transm. 107, 1165-1173.
Le Moine C and B Bloch (1991) Rat striatal and mesencephalic
neurons contain the long isoform of the D2 dopamine receptor
mRNA. Brain Res. Mol. Brain Res. 10, 283-289.
Ling ZD, HC Robie, CW Tong and PM Carvey (1999) Both the
antioxidant and D3 agonist actions of pramipexole mediate its
neuroprotective actions in mesencephalic cultures. J. Pharmacol.
Exp. Ther. 289, 202-210.
Ling ZD, CW Tong and PM Carvey (1998) Partial purification of a
pramipexole-induced trophic activity directed at dopamine neurons in ventral mesencephalic cultures. Brain Res. 791, 137-145.
Marek K, J Seibyl, I Shoulson, R Holloway, K Kieburtz, M
McDermott, C Kamp, A Shinaman, S Fahn, A Lang, W Weiner
and M Welsh (2002) Dopamine transporter brain imaging to
assess the effects of pramipexole vs levodopa on Parkinson disease progression. JAMA 287, 1653-1661.
Martel F, C Calhau and I Azevedo (2000) Characterization of the
transport of the organic cation [3H]MPP+ in human intestinal
epithelial (Caco-2) cells. Naunyn Schmiedebergs Arch.
Pharmacol. 361, 505-513.
Masserano JM, L Gong, H Kulaga, I Baker and RJ Wyatt (1996)
Dopamine induces apoptotic cell death of a catecholaminergic
cell line derived from the central nervous system. Mol.
Pharmacol. 50, 1309-1315.
Matecka D, RB Rothman, L Radesca, BR de Costa, CM Dersch, JS
Partilla, A Pert, JR Glowa, FH Wojnicki and KC Rice (1996)
Development of novel, potent, and selective dopamine reuptake
inhibitors through alteration of the piperazine ring of 1-[2(diphenylmethoxy)ethyl]-and
1-[2-[bis(4fluorophenyl)methoxy]ethyl]-4- (3-phenylpropyl)piperazines
(GBR 12935 and GBR 12909). J. Med. Chem. 39, 4704-4716.
Matsuoka I, N Mizuno and K Kurihara (1989) Cholinergic differentiation of clonal rat pheochromocytoma cells (PC12) induced
by retinoic acid: increase of choline acetyltransferase activity
and decrease of tyrosine hydroxylase activity. Brain Res. 502,
53-60.
598
McNaught KS, U Thull, PA Carrupt, C Altomare, S Cellamare, A

Carotti, B Testa, P Jenner and CD Marsden (1996) Nigral cell
loss produced by infusion of isoquinoline derivatives structurally related to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.
Neurodegeneration 5, 265-274.
Muralikrishnan D and KP Mohanakumar (1998) Neuroprotection
by bromocriptine against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced neurotoxicity in mice. FASEB J. 12, 905912.
Murer MG, G Dziewczapolski, LB Menalled, MC Garcia, Y Agid,
O Gershanik and R Raisman-Vozari (1998) Chronic levodopa is
not toxic for remaining dopamine neurons, but instead promotes
their recovery, in rats with moderate nigrostriatal lesions. Ann.
Neurol. 43, 561-575.
Oyarce AM and PJ Fleming (1991) Multiple forms of human
dopamine beta-hydroxylase in SH-SY5Y neuroblastoma cells.
Arch. Biochem. Biophys. 290, 503-510.
Park CW, HS Lee and YS Kim (1998) Mechanism of MPP(+)induced cytotoxicity in human neuroblastoma SH-SY5Y. J.
Toxicol. Sci. 23 Suppl 2, 184-188.
Pennypacker KR, DM Kuhn and ML Billingsley (1989) Changes in
expression of tyrosine hydroxylase immunoreactivity in human
SMS-KCNR neuroblastoma following retinoic acid or phorbol
ester-induced differentiation. Brain Res. Mol. Brain Res. 5, 251258.
Przedborski S, V Jackson-Lewis, R Djaldetti, G Liberatore, M Vila,
S Vukosavic and G Almer (2000) The parkinsonian toxin
MPTP:action and mechanism. Restorative Neurol. Neurosci. 16,
135-142.
Ramirez AD, SK Wong and FS Menniti (2003) Pramipexole
inhibits MPTP toxicity in mice by dopamine D3 receptor dependent and independent mechanisms. Eur. J. Pharmacol. 475, 29-35.
Richards ML and W Sadee (1986) Human neuroblastoma cell lines
as models of catechol uptake. Brain Res. 384, 132-137.
Ross RA, BA Spengler and JL Biedler (1983) Coordinate morphological and biochemical interconversion of human neuroblastoma cells. J. Natl. Cancer Inst. 71, 741-747.
Sheehan JP, PE Palmer, GA Helm and JB Tuttle (1997) MPP+
induced apoptotic cell death in SH-SY5Y neuroblastoma cells:
an electron microscope study. J. Neurosci. Res. 48, 226-237.
Simantov R, E Blinder, T Ratovitski, M Tauber, M Gabbay and S
Porat (1996) Dopamine-induced apoptosis in human neuronal
cells: inhibition by nucleic acids antisense to the dopamine trans-
porter. Neuroscience 74, 39-50.

Song X, S Perkins, BS Jortner and M Ehrich (1997) Cytotoxic
effects of MPTP on SH-SY5Y human neuroblastoma cells.
Neurotoxicology 18, 341-353.
Spina MB, SP Squinto, J Miller, RM Lindsay and C Hyman (1992)
Brain-derived neurotrophic factor protects dopamine neurons
against 6- hydroxydopamine and N-methyl-4-phenylpyridinium
N
ion toxicity: involvement of the glutathione system [see comments]. J. Neurochem. 59, 99-106.
Stokes AH, DY Lewis, LH Lash, WG Jerome, III, KW Grant, M
Aschner and KE Vrana (2000) Dopamine toxicity in neuroblastoma cells: role of glutathione depletion by L-BSO and apoptosis. Brain Res. 858, 1-8.
Storch A, K Burkhardt, AC Ludolph and J Schwarz (2000a)
Protective effects of riluzole on dopamine neurons: involvement
of oxidative stress and cellular energy metabolism. J.
Neurochem. 75, 2259-2269.
Storch A, A Kaftan, K Burkhardt and J Schwarz (2000b) 6Hydroxydopamine toxicity towards human SH-SY5Y dopaminergic neuroblastoma cells: independent of mitochondrial energy
metabolism. J. Neural Transm. 107, 281-293.
Veech GA, J Dennis, PM Keeney, CP Fall, RH Swerdlow, WD
Parker Jr and JP Bennett Jr (2000) Disrupted mitochondrial electron transport function increases expression of anti-apoptotic bcl2 and bcl-X(L) proteins in SH-SY5Y neuroblastoma and in
Parkinson disease cybrid cells through oxidative stress. J.
Neurosci. Res. 61, 693-700.
Willets JM, DG Lambert, J Lunec and HR Griffiths (1995) Studies
on the neurotoxicity of 6,7-dihydroxy-1-methyl-1,2,3,4- tetrahydroisoquinoline (salsolinol) in SH-SY5Y cells. Eur. J.
Pharmacol. 293, 319-326.
Zapata A and TS Shippenberg (2002) D3 receptor ligands modulate
extracellular dopamine clearance in the nucleus accumbens. J.
Neurochem. 81, 1035-1042.
Zapata A, JM Witkin and TS Shippenberg (2001) Selective D3
receptor agonist effects of (+)-PD 128907 on dialysate dopamine
at low doses. Neuropharmacology 41, 351-359.
Zou L, J Xu, J Jankovic, Y He, SH Appel and W Le (2000)
Pramipexole inhibits lipid peroxidation and reduces injury in the
substantia nigra induced by the dopaminergic neurotoxin 1methyl-4- phenyl-1,2,3,6-tetrahydropyridine in C57BL/6 mice.
Neurosci. Lett. 281, 167-170.

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Neurotoxicity Research, 2004, VOL. 5(8). pp.

Terminally Differentiated SH-SY5Y Cells Provide a Model

We characterized undifferentiated (UN) and three

neuroprotective effects of DA agonists in this model

S.P. PRESGRAVES et al.

1983). There are a number of related compounds to

MATERIALS AND METHODS

SH-SY5Y CELLS AS A MODEL TO STUDY NEUROPROTECTION

Cytotoxic Effects of MPP+ and DA on SH-SY5Y

MTT Assay for Cell Viability

Lactate Dehydrogenase Assay for Cell Viability

S.P. PRESGRAVES et al.

At different time points,

SH-SY5Y CELLS AS A MODEL TO STUDY NEUROPROTECTION

(RT) then permeabilized by treatment with 0.1%

buffer (TBS buffer with 00.05%

S.P. PRESGRAVES et al.

D1 or D2 receptors) and various concentrations of MPP+

are much higher but still predominantly observed in the

SH-SY5Y CELLS AS A MODEL TO STUDY NEUROPROTECTION

S.P. PRESGRAVES et al.

al., 2001). The Km values were also similar to that

preparations but the Km was higher. Direct comparison

SH-SY5Y CELLS AS A MODEL TO STUDY NEUROPROTECTION

than for [3H]DA uptake, they were not statistically different.

MPP+ and DA Neurotoxicity

S.P. PRESGRAVES et al.

FIGURE 4 [3H]DA or [3H]MPP+ uptake in synaptosomes prepared

varying concentrations of DA (0 to 500 M) were

SH-SY5Y CELLS AS A MODEL TO STUDY NEUROPROTECTION

C ell death with DA at 48 hrs

Percent LDH Release

Percent Cell Death

A 100MPP+ Toxicity at 0.03 mM

Percent Cell Death

10 50 100 150 250 500

M PP+ Toxicity at 0.3 mM

Percent LDH Release

Percent Cell Death

C ell Death with DA at 72 hrs

10 50 100 150 250 500

S.P. PRESGRAVES et al.

Effect of Transporter Inhibiitors on Neurotoxicity

differentiated cells exhibited greater sensitivity than

SH-SY5Y CELLS AS A MODEL TO STUDY NEUROPROTECTION

(FIG. 10-A) or mitochondrial dysfunction (FIG. 10-B)

cells at the lowest concentration but provided modest

S.P. PRESGRAVES et al.

FIGURE 8 GBR 12909 blocks neurotoxic effects of MPP+ in

presence or absence of a D2/D3/D4 antagonist (spiperone), D3 preferring antagonist (U99194A), or the D1

SH-SY5Y CELLS AS A MODEL TO STUDY NEUROPROTECTION

S.P. PRESGRAVES et al.

SH-SY5Y CELLS AS A MODEL TO STUDY NEUROPROTECTION

This is in contrast to undifferentiated SH-SY5Y cells

cell loss measured with this protocol is less than that

S.P. PRESGRAVES et al.

(Simantov et al., 1996; Iida et al., 1999; Jones et al.,

mesencephalic dopaminergic neurons. J. Neurosci. 22, 26372649.

SH-SY5Y CELLS AS A MODEL TO STUDY NEUROPROTECTION

methyl-4-phenylpyridinium-induced apoptotic death in human

S.P. PRESGRAVES et al.

McNaught KS, U Thull, PA Carrupt, C Altomare, S Cellamare, A

porter. Neuroscience 74, 39-50.