Journal of Neurochemistry
Lippincott Williams & Wilkins, Inc., Philadelphia
2000 International Society for Neurochemistry
Protective Effects of Riluzole on Dopamine Neurons:
Involvement of Oxidative Stress and Cellular
Energy Metabolism
Alexander Storch, Katrin Burkhardt, Albert C. Ludolph, and Johannes Schwarz
Department of Neurology, University of Ulm Medical School, Ulm, Germany
Abstract: Riluzole is neuroprotective in patients with
amyotrophic lateral sclerosis and may also protect dopa-
mine (DA) neurons in Parkinsons disease. We examined
the neuroprotective potential of riluzole on DA neurons
using primary rat mesencephalic cultures and human
dopaminergic neuroblastoma SH-SY5Y cells. Riluzole
(up to 10 M) alone affected neither the survival of DA
neurons in primary cultures nor the growth of SH-SY5Y
cells after up to 72 h. Riluzole (110 M) dose-depen-
dently reduced DA cell loss caused by exposure to MPP
in both types of cultures. These protective effects were
accompanied by a dose-dependent decrease of intracel-
lular ATP depletion caused by MPP (30 300 M) in
SH-SY5Y cells without affecting intracellular net NADH
content, suggesting a reduction of cellular ATP consump-
tion rather than normalization of mitochondrial ATP pro-
duction. Riluzole (110 M) also attenuated oxidative
injury in both cell types induced by exposure to L-DOPA
and 6-hydroxydopamine, respectively. Consistent with
its antioxidative effects, riluzole reduced lipid peroxida-
tion induced by Fe3 and L-DOPA in primary mesence-
phalic cultures. Riluzole (10 M) did not alter high-affinity
uptake of either DA or MPP. However, in the same cell
systems, riluzole induced neuronal and glial cell death
with concentrations higher than those needed for maxi-
mal protective effects (100 M). These data demon-
strate that riluzole has protective effects on DA neurons in
vitro against neuronal injuries induced by (a) impairment
of cellular energy metabolism and/or (b) oxidative stress.
These results provide further impetus to explore the neu-
roprotective potential of riluzole in Parkinsons disease.
Key Words: RiluzoleParkinsons disease1-Methyl-
4-phenylpyridinium 6-HydroxydopamineDopamine
neuronsNeuroprotection.
J. Neurochem. 75, 2259 2269 (2000).
Riluzole (2-amino-6-trifluoromethoxybenzothiazole)
can slow the progression of disease in patients with
amyotrophic lateral sclerosis and has accordingly been
approved for the treatment of this disorder in several
countries (Bensimon et al., 1994; Lacomblez et al.,
1996). Present clinical studies aim to explore the protec-
tive potential of this compound in other disorders, such
2259
as Parkinsons disease (PD). Riluzole is neuroprotective
in animal models of acute and chronic neurodegenerative
disease (Stutzmann and Doble, 1994; Mary et al., 1995;
McIntosh et al., 1996; Palfi et al., 1997; Kanthasamy
et al., 1999), including rat and primate models of PD
(Benazzouz et al., 1995; Barneoud et al., 1996; Bezard
et al., 1998; Boireau et al., 2000).
The neuroprotective mechanism of riluzole was ini-
tially attributed to its inhibitory effect on glutamatergic
neurotransmission (Martin et al., 1993; MacIver et al.,
1996) and the resultant excitotoxic neuronal injury (Es-
tevez et al., 1995; Mary et al., 1995; Doble, 1999).
However, the pharmacology of riluzole has turned out to
be more complex: (a) Riluzole has been reported to
inhibit multiple ion channels such as glutamate-gated
channels (Debono et al., 1993; Hubert et al., 1994),
voltage-gated channels (Benoit and Escande, 1991; He-
bert et al., 1994; Song et al., 1997), and volume-sensitive
chloride channels (Bausch and Roy, 1996). Blocking
these ion channels may result in protection against exci-
totoxicity, but decreased ion fluxes through these chan-
nels also lead to lower transport rates of ion pumps, in
particular the Na,K-ATPase, and subsequent reduced
cellular energy demand. Because a large part of cellular
energy in nerve cells is needed to maintain ion gradients
across the cell membrane (up to 70% of cellular ATP)
(Pedersen and Carafoli, 1987), this reduction may result
in neuroprotection, at least in conditions associated with
deficient cellular energy supply (Urenjak and Obreno-
Received March 7, 2000; revised manuscript received June 29, 2000;
accepted July 14, 2000.
Address correspondence and reprint requests to Dr. A. Storch at
Department of Neurology, University of Ulm Medical School, Oberer
Eselsberg 45, 89081 Ulm, Germany. E-mail: alexander.storch@
medizin.uni-ulm.de
The present address of Dr. J. Schwarz is Division of Biology,
California Institute of Technology, Pasadena, CA, U.S.A.
Abbreviations used: DA, dopamine; DAT, dopamine transporter;
MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;
6-OHDA, 6-hydroxydopamine; PD, Parkinsons disease; TBARS, thio-
barbituric acid-reactive substances; TD50, half-maximal toxic dose;
TH, tyrosine hydroxylase; THir, tyrosine hydroxylase-immunoreactive.
2260 A. STORCH ET AL.
vitch, 1998). (b) Recently, Koh et al. (1999) showed
antioxidant effects of riluzole on cultured cortical neu-
rons in vitro. (c) Furthermore, riluzole stimulates the
production of trophic activity for motoneurons in culture
by spinal astrocytes (Peluffo et al., 1997). (d) In contrast,
at higher concentrations (100 M) riluzole induces
apoptotic cell death in primary cortical cultures in a
caspase-sensitive manner (Koh et al., 1999).
Impairment of cellular energy metabolism and/or ox-
idative stress are most likely involved in the patho-
mechanism of selective degeneration of dopamine (DA)
neurons in PD (for review, see Dunnett and Bjorklund,
1999): Reduction of mitochondrial NADH-Q reductase
(mitochondrial complex I) activity has been demon-
strated in substantia nigra, nonnigral brain areas, plate-
lets, muscle, and fibroblasts of patients with PD (Mizuno
et al., 1989; Schapira et al., 1990; Krige et al., 1992).
Furthermore, a reduction of -ketoglutarate dehydroge-
nase complex activity of the tricarboxylic acid cycle has
also been observed in PD (Mizuno et al., 1994). On the
other hand, there are several arguments supporting a role
for oxidative stress in the pathochemical substrate of PD
(for review, see Cadet and Brannock, 1998): For exam-
ple, levels of lipid peroxidation products are significantly
elevated in brain from PD patients (Dexter et al., 1989),
and iron metabolism is reported to be abnormal in the
basal ganglia of individuals suffering from PD, with
increased levels of Fe3 in the substantia nigra pars
compacta (Riederer et al., 1989). In addition to the
changes in iron content, levels of several free radical
scavenging systems, including the glutathione system,
are abnormal in PD brains (for review, see Cadet and
Brannock, 1998). Most approaches for neuroprotective
therapies in PD were aimed at the reduction of oxidative
stress, for example, using two putative antioxidative
agents, the monoamine oxidase B inhibitor deprenyl and
vitamin E, respectively, but both strategies failed to show
significant neuroprotective effects in PD patients (Shoul-
son, 1998).
Although riluzole has been demonstrated to be neuro-
protective in animal models of PD produced by selective
dopaminergic neurotoxins, such as 1-methyl-4-phenyl-
1,2,3,6-tetrahydropyridine (MPTP) and 6-hydroydopam-
ine (6-OHDA) in primates and rats, respectively (Benaz-
zouz et al., 1995; Barneoud et al., 1996; Bezard et al.,
1998; Boireau et al., 2000), the mechanism of the pro-
tective effects against these toxins on DA neurons has to
be determined. However, there is no interference of
riluzole with MPTP metabolism in vivo (Boireau et al.,
2000). MPTP causes a parkinson-like syndrome in hu-
man and nonhuman primates, most likely secondary to
production of free radicals through its biotransformation
by monoamine oxidase B (Gerlach et al., 1991; Przed-
borski and Jackson-Lewis, 1998) and subsequent inhibi-
tion of mitochondrial complex I by its metabolite,
1-methyl-4-phenylpyridinium ion (MPP) (Nicklas
et al., 1985; for review, see Gerlach et al., 1991; Przed-
borski and Jackson-Lewis, 1998). The mechanism of
selective dopaminergic toxicity of 6-OHDA in vivo is
J. Neurochem., Vol. 75, No. 6, 2000
not fully understood, but the main mechanism most
likely involves production of free radicals and quinones,
as demonstrated also in vitro (Tiffany-Castiglioni et al.,
1982; Michel and Hefti, 1990; Abad et al., 1995; Cadet
and Brannock, 1998; Storch et al., 2000c). Consequently,
the mechanism of protective effects of riluzole on DA
neurons in animal models of PD, as well as in PD
patients, should include normalization of impaired cel-
lular energy metabolism and/or antioxidative effects.
To test these hypotheses, we set out to determine the
neuroprotective spectrum of riluzole on primary DA
neurons from rat and on human dopaminergic neuroblas-
toma SH-SY5Y cells, using selective dopaminergic neu-
rotoxins causing either impairment of cellular energy
metabolism by inhibition of mitochondrial complex I
(MPP) or oxidative stress (L-DOPA and 6-OHDA).
MATERIALS AND METHODS
Materials
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT), L-DOPA, 6-OHDA, Fe(III)Cl3, and the nucleo-
tide standards of the highest grade available were purchased
from Sigma (St. Louis, MO, U.S.A.). MPP iodide was ob-
tained from Research Biochemicals International (Natick, MA,
U.S.A.). [3H]DA (48 Ci/mmol) was purchased from Amersham
International (Braunschweig, Germany), and [3H]MPP (30
Ci/mmol) was from NEN DuPont (Koln, Germany). Ammo-
nium phosphate and HPLC-grade methanol were purchased
from Merck (Darmstadt, Germany). Riluzole was kindly pro-
vided by Dr. Wilhelm Fischer from Rhone Poulenc-Rorer
(Koln). Treatments of cultures with riluzole and medium con-
ditioning were performed with 10 mM stock solutions of ri-
luzole prepared by dissolving in 0.01 M HCl. All other chem-
icals were of analytical grade.
Cultures
Primary cultures. Primary cultures from rat rostral mesen-
cephalic tegmentum were prepared as previously described
(Storch et al., 2000a). In brief, the rostral mesencephalic teg-
mentum from embryonic day 14.5 rat embryos (Wistar;
Charles-River, Braunschweig) was dissociated using trypsin,
DNase, and mechanical trituration. The cells were plated out at
125,000 viable cells/cm2 on poly-L-lysine-coated 48- or 24-
well plates (for uptake studies) in complete medium containing
Dulbeccos modified Eagles medium (high glucose)/F-12 mix-
ture (1:1), 1% penicillin/streptomycin (Sigma), and 10% fetal
calf serum (PAA, Colbe, Germany). Cultures were incubated at
37C in a humidified atmosphere of 5% CO2 in air. The culture
medium was changed 24 h after plating. Striatal cultures were
generated using essentially the same procedure except that the
lateral ganglionic eminence was dissected from embryonic day
14.5 rat embryos.
SH-SY5Y cell system. The human dopaminergic neuroblas-
toma SH-SY5Y cell line (Ross and Biedler, 1985) was obtained
from the Deutsche Sammlung Mikroorganismen (Braun-
schweig). The cells were cultured in Dulbeccos minimum
essential medium (GIBCO, Eggenstein, Germany) supple-
mented with 15% fetal bovine serum in a water-jacketed incu-
bator at 37C in a humidified atmosphere of 10% CO2/90% air.
The cells were routinely harvested by trypsinization (0.05%
trypsin) when the cells approached the subconfluent stage and
plated in 25-cm2 culture flasks.
 PROTECTION OF DOPAMINE NEURONS BY RILUZOLE
Treatment paradigms
Primary cultures. Primary cultures were treated after 4 days
in culture with various concentrations of MPP, L-DOPA,
6-OHDA, and Fe(III)Cl3 alone or in combination with riluzole
for up to 48 h. For experiments with MPP, a low-energy
medium was used according to the procedure of Kaufman and
Barrett (1983) containing only 50 mM D-glucose. Then, the
cultures were further processed for immunocytochemical stud-
ies or determination of lipid peroxidation.
Conditioned medium experiments. Striatal cultures were ex-
posed after 5 days in culture to freshly prepared riluzole (at
various concentrations) for 36 h. Then the cultures were
washed three times with defined medium consisting of Dulbec-
cos modified Eagles medium (high glucose)/F-12 mixture
(1:1) supplemented with transferrin (100 g/ml), insulin (25
g/ml), progesterone (20 nM), putrescine (62 M), sodium
selenite (30 nM), and 1% penicillin/streptomycin (all supple-
ments from Sigma) and further incubated with the same me-
dium. The medium was removed after 24 h and centrifuged at
1,800 g for 15 min, and the supernatant was used immediately
or stored at 20C for not more than 4 weeks.
SH-SY5Y cells. For toxicological studies the cells were
seeded in 96-well plates at a density of 12,500 cells per well in
200 l of medium (different seeding densities were optimized
at the beginning of the experiments; data not shown). The
cultures were grown for 48 h, and then 50 l of medium
containing various concentrations of MPP, L-DOPA, or
6-OHDA alone or in combination with freshly prepared ri-
luzole was added. After an incubation period of up to 72 h, cell
viability was assessed using the MTT assay. For determination
of intracellular net contents of adenine and pyridine nucleo-
tides, SH-SY5Y cells were seeded in 25-cm2 culture flasks or
10-mm-diameter Petri dishes at a density of 7.5 105 cells/
cm2 and grown for 48 h. Then, the culture medium was
changed to one containing various concentrations of MPP
with or without riluzole, and the cells were incubated for up to
48 h.
Immunocytochemical and biochemical techniques
Quantification of dopaminergic cell survival in primary cul-
tures. Following treatment, the cultures were fixed using 3.7%
paraformaldehyde and then immunocytochemically processed
for tyrosine hydroxylase (TH) using a monoclonal antibody
against rat TH (Boehringer Mannheim, Mannheim, Germany)
and nickel enhancement. The numbers of TH-immunoreactive
(THir) cells were assessed by an investigator blinded to treat-
ment history using a Zeiss inverted microscope, counting black
cell bodies of the whole well surface area. Based on the tissue
dissection procedure and the absence of immunoreactivity for
the noradrenergic neuron marker DA -hydroxylase in sister
cultures, it is likely that THir cells in our cultures were phe-
notypically DA neurons.
MTT assay. After incubation for up to 72 h, 30 l of MTT
reagent (0.5 mg/ml MTT in phosphate-buffered saline contain-
ing 10 mM HEPES) was added to each well and incubated at
37C for 2 h. The medium was aspirated from each well, and
the culture plate was dried at 37C for 1 h. The resulting
formazan dye was extracted with 100 l of acidic isopropanol,
and the absorbancy was measured spectrophotometrically with
a computer-operated immunoreader (Molecular Dynamics,
Germany) at a wavelength of 570 nm with reference at 630 nm.
Wells without cells were used as blanks, and their values were
subtracted as background from each sample.
2261
Measurement of intracellular adenine and pyridine levels by
HPLC. The intracellular content of adenine nucleotides and
NADH was measured as previously described (Storch et al.,
1999). In brief, after treatment with the substance of interest,
cells were harvested using trypsin/EDTA, cell count was mea-
sured using the trypan blue exclusion method, and adenine
nucleotides were extracted using 1 M perchloric acid. After
centrifugation (13,000 rpm for 10 min) to separate the dena-
tured protein, to 600 l of the supernatant, 400 l of ice-cold
0.5 M KHCO3 was added dropwise for neutralization. After
centrifugation at the condition mentioned above, 100 l of the
supernatant was injected into the HPLC apparatus using a loop
valve. The intracellular content of NADH was measured using
an alkaline extraction: Cells were rinsed with ice-cold phos-
phate-buffered saline and then scraped into liquid nitrogen.
After thawing, 50 l of the suspension was stored at 80C for
later protein content determination, and 500 l was treated with
500 l of ice-cold 0.5 M KOH solution and immediately
deproteinized by shaking. After 5 min, 1 ml of ice-cold water
was added, and the resulting solution was placed on a Centricon
membrane (30,000-Da cutoff; Amicon, Lexington, MA,
U.S.A.) and centrifuged at 2,500 rpm for 10 min. Then 500 l
of the ultrafiltered solution was neutralized by adding 75 l of
ice-cold 1 M KH2PO4 solution, and the resulting solution was
injected into the HPLC apparatus. Nucleotides were separated
using a methanol gradient reverse-phase method. A Gynkotec
(Munich, Germany) model 480 pump with solvent programmer
was used, and the separation was performed at room tempera-
ture with a 5-m (particle size) Supelcosil LC-18 (25 cm 4.6
mm i.d.; Supelco, Belafonte, PA, U.S.A.) reversed-phase col-
umn and UV (254 nm) and fluorescence detection (excitation at
340 nm, emission at 460 nm). The mobile phase consisted of
100 mM ammonium phosphate solution (pH 6.0) containing
increasing amounts of methanol. The gradient was started 4
min after injection of the sample and got up to 10% (vol/vol) of
methanol in 21 min. For detection of the reduced pyridine
nucleotides, the assay was kept isocratic at 7% methanol. The
flow rate was 1.3 ml/min. Assignment of nucleotide peaks and
calculation of absolute concentration were obtained by com-
parison with external standards of known nucleotide composi-
tion and concentration. The limits of detection were 7, 11, 4,
and 15 pmol per sample for ATP, ADP, NAD, and NADH,
respectively.
Measurement of lipid peroxidation. Lipid peroxides were
quantified using the thiobarbituric acid-reactive substances
(TBARS) assay according to the method of Ohkawa et al.
(1979). In brief, mesencephalic cultures grown in 48-well
plates were lysed with 300 l of 2% sodium dodecyl sulfate for
30 min. The protein concentration was quantitatively measured
by the method of Bradford (1976) using the Bio-Rad determi-
nation kit and bovine serum albumin as the standard. The
lysates (250 l) were added serially with 12.5 l of butylated
hydroxytoluene (4% in ethanol), 250 l of phosphotungstic
acid (10% in 0.5 M sulfuric acid), and 125 l of thiobarbituric
acid (0.7%). The mixtures were incubated at 95C for 60 min,
and then 300 l of n-butanol was added to extract the TBARS.
After centrifugation at 4,000 rpm for 10 min, the supernatant
was collected (samples from three or four culture wells were
pooled), and the amounts of TBARS were measured at 535 nm
using a spectral photometer. TBARS were scaled using a mix-
ture of 1 mM tetrahydroxypropane in 1% sulfuric acid and
expressed in nanomoles per milligram of protein.
Neurotransmitter uptake studies. The influence of riluzole on
high-affinity uptake of dopamine or MPP by the DA trans-
J. Neurochem., Vol. 75, No. 6, 2000
2262 A. STORCH ET AL.
porter (DAT) was evaluated by measuring the uptake of the
respective tritiated substance on primary mesencephalic cul-
tures according to the technique of Storch et al. (2000a) and
Reinhard et al. (1987). After preincubation of riluzole (10 M)
for 30 min in incubation buffer containing 100 M pargyline
and 1 mM ascorbate (and, for determination of nonspecific
uptake, 3 M GBR12909) at 37C, 50 nM [3H]DA or
[3H]MPP was added in the absence and presence of riluzole
(10 M) for 15 min at 37C. Uptake was stopped by washing
the dishes with cold phosphate-buffered saline, and the remain-
ing radioactivity in the cell lysate was measured using liquid
scintillation counting. Specific uptake was defined as the dif-
ference between the uptake measured in the absence (total) and
the uptake measured in the presence (nonspecific) of
GBR12909.
Statistical analysis
Half-maximal toxic dose (TD50) values were calculated by
nonlinear regression analysis using the iterative curve-fitting
program Origin (version 5.0; MicroCal Software). Results were
expressed as mean SEM values of at least three independent
experiments and compared using paired or unpaired t test.
RESULTS
Effects of riluzole on survival of DA neurons
Before investigating the effects of riluzole on selective
dopaminergic neurotoxicity, we determined the effects of
riluzole on cell viability of DA neurons in vitro. Riluzole
did not influence the morphology and cell count of THir
cells in mesencephalic cultures at concentrations up to 10
M after 48 h, whereas 100 M significantly reduced
THir cell survival (Figs. 1 and 2). Based on gross mi-
croscopic inspection of the cultures, these high concen-
trations of riluzole killed all cell types, including glial
cells. Furthermore, riluzole showed no effects on cell
viability in SH-SY5Y cells measured with the MTT
assay with concentrations up to 10 M after a treatment
period of up to 72 h, whereas 100 M riluzole signifi-
cantly reduced cell viability to 77 6% of control ( p
0.05).
Riluzole protects DA neurons against
MPP-induced toxicity
MPP caused a dose-related decrease of THir cells in
4-day-old mesencephalic cultures from rats, with a cal-
culated TD50 obtained from the toxicity curve by non-
linear regression analysis of 2.6 0.8 M after 48 h of
incubation (Fig. 3A). The remaining THir cells showed
fewer and shorter neurites compared with cells without
MPP treatment (see Fig. 1). These observations are
consistent with the data reported in the literature (Michel
et al., 1990). A concentration of 1 M riluzole, which did
not affect THir neuron survival when administered alone,
shifted the MPP survival curve significantly to the right
(Fig. 3A). Thus, in the presence of 1 M riluzole, the
0.310 M concentrations of MPP exhibited signifi- et al., 2000b). Using essentially the same protocol, we
cantly higher THir cell counts relative to cultures treated
with MPP alone ( p 0.05). Furthermore, the THir
cells appeared healthier and exhibited longer neurites
(Fig. 1). The calculated TD50 value obtained from the
toxicity curves by nonlinear regression analysis in-
J. Neurochem., Vol. 75, No. 6, 2000
creased from 2.6 0.8 M for MPP alone up to 15.5
2.4 M for MPP combined with riluzole (signifi-
cantly different with p 0.01).
As reported by Peluffo et al. (1997), riluzole has
trophic effects on primary motoneurons in culture by
stimulating trophic activity of astrocytes. To examine
whether a glial cell-dependent trophic effect of riluzole is
responsible for its protection against MPP toxicity,
conditioned-medium transfer studies were performed.
Medium collected from mixed striatal cultures (contain-
ing glial and neuronal cells) previously exposed to var-
ious concentrations of riluzole for 36 h did not change
THir cell counts in recipient mesencephalic cultures (Fig.
4). Using the approximate TD50 of MPP (3 M), the
effects of striatal medium conditioned with various con-
centrations of riluzole on MPP-induced toxicity in mes-
encephalic cultures was determined. As shown in Fig. 4,
riluzole-conditioned medium did not significantly alter
dopaminergic toxicity induced by MPP in vitro.
In previous studies using the MTT assay we were able
to show a time- and dose-dependent cytotoxicity of
MPP against human dopaminergic neuroblastoma SH-
SY5Y cells with slow onset and time course (Storch
et al., 2000b,c). As shown in Fig. 3B, riluzole signifi-
cantly shifted the MPP toxicity curve in a dose-depen-
dent manner to the right, resulting in an increase of the
TD50 value from 76.9 8.4 M for MPP alone to
264.2 30.9 and 920 M for MPP in combination
with riluzole at concentrations of 1 and 10 M, respec-
tively ( p 0.05). Despite the fact that 100 M riluzole
induced a mild loss of cell viability (see above), this
concentration caused complete protection against MPP
with concentrations up to 300 M (data not shown).
Riluzole normalizes cellular energy supply altered
by MPP
As a step in investigating possible mechanisms of
riluzole-induced cytoprotection, we tested the effects of
riluzole on cellular energy supply altered by MPP in
SH-SY5Y cells by measuring intracellular net ATP lev-
els using an HPLC-based method. After a treatment
period of 48 h, the control value for intracellular (cyto-
solic and mitochondrial) net ATP content was 1.33
0.25 nmol of ATP per 106 cells. This value is in the
same range as those measured in previous studies using
the same cell type (Storch et al., 2000b) as well as in
isolated hepatocytes and human embryonic kidney 293
cells (Siess and Wieland, 1976; Di Monte et al., 1986;
Storch et al., 1999). As shown previously, MPP caused
a time- and dose-dependent decrease in net intracellular
ATP content with an IC50 value of 66.0 15.7 M after
48 h, which consistently precedes cell death (Storch
were able to show that riluzole at concentrations that did
not affect intracellular ATP levels when administered
alone (1 and 10 M) dose-dependently reduces cellular
ATP depletion caused by MPP in SH-SY5Y cells (Fig.
5A). The potency of riluzole for this effect is in the same
 PROTECTION OF DOPAMINE NEURONS BY RILUZOLE 2263

FIG. 1. Photomicrographs demonstrate the effects of riluzole on dopaminergic toxicity induced by MPP after 48 h or L-DOPA after 24 h
in mesencephalic cultures. A: THir neurons in a control culture at day 6 in vitro. BI: THir neurons in sister cultures exposed to (B) 1 M
riluzole, (C) 10 M riluzole, (D) 3 M MPP, (E) 3 M MPP plus 1 M riluzole, (F) 3 M MPP plus 10 M riluzole, (G) 30 M L-DOPA,
(H) 30 M L-DOPA plus 1 M riluzole, or (I) 30 M L-DOPA plus 10 M riluzole. Bar 30 m.

range as that estimated for the protection against MPP-
induced cell death (compare with Fig. 3B).
Because MPP is known to inhibit oxidation of
NADH by blocking mitochondrial complex I activity
(Nicklas et al., 1985; for review, see Gerlach et al.,
1991; Przedborski and Jackson-Lewis, 1998), MPP
causes a fast and significant increase of intracellular
net NADH content in SH-SY5Y cells with a maximum
after 12 h (Storch et al., 2000b). To study whether
riluzole alters the potency of MPP to inhibit complex
I activity, the effects of riluzole on MPP-induced
changes of intracellular NADH content in SH-SY5Y
cells were measured using an HPLC-based method.
The control value of intracellular (cytosolic and
mitochondrial) net NADH (1.12 0.12 nmol of
NADH/mg of protein) was in the same range as that
measured in human embryonic kidney 293 cells (8.3
0.7 nmol of NADH/mg of protein) (Storch et al.,
1999). MPP caused an increase to 251 19% of
control after 12 h ( p 0.05). As shown in Fig. 5B,
riluzole did not alter the MPP-induced changes of
intracellular net NADH content significantly.

J. Neurochem., Vol. 75, No. 6, 2000

2264 A. STORCH ET AL.

SH-SY5Y cells, 6-OHDA showed a TD50 value of 24.6

3.2 M after 24 h (Storch et al., 2000c). As shown in
Fig. 7B, riluzole at concentrations of 1100 M signif-
icantly reduced 6-OHDA-induced toxicity in SH-SY5Y
cells.
Riluzole decreases lipid peroxidation caused by
iron-III and L-DOPA in mesencephalic cultures
Because L-DOPA (for review, see Ling et al., 1996;
Storch et al., 2000a) and 6-OHDA (Tiffany-Castiglioni
et al., 1982; Michel and Hefti, 1990; Abad et al., 1995;
Storch et al., 2000c) are toxic to dopaminergic cells
mainly by generation of free radicals, we investigated the
antioxidative effects of riluzole by measuring lipid per-
oxidation using the TBARS assay. Control levels of lipid

FIG. 2. Effects of riluzole on survival of DA neurons in mesen-

cephalic cultures. The cells were cultured for 4 days before
treatment with various concentrations of riluzole for 24 or 48 h.
Survival of THir neurons is expressed as a percentage of the
untreated control. Control values were 562 126 and 497 141
THir cells per well for the 24- and 48-h treatment period, respec-
tively. Data are mean SEM (bars) values of at least three
independent experiments. **p 0.01 compared with untreated
control.

Riluzole does not influence uptake of DA or MPP

To rule out uptake blockade as a mechanism of pro-
tection against MPP and 6-OHDA toxicity, the uptake
of [3H]DA and [3H]MPP, respectively, was measured
in the presence and absence of riluzole (10 M) in
primary mesencephalic cultures. As shown in Fig. 6,
riluzole at a concentration that is sufficient to produce
protection against MPP toxicity did not significantly
block both DA and MPP high-affinity uptake by the
DAT.
Riluzole protects DA neurons against L-DOPA- and
6-OHDA-induced toxicity
As reported previously, L-DOPA caused a dose-related
decrease in number of THir cells in 4-day-old mesence-
phalic cultures from rats, with a calculated TD50 value of
21.3 1.4 M after 24 h (Ling et al., 1996; Storch et al.,
2000a). The remaining THir cells showed fewer and
shorter neurites compared with cells without L-DOPA
treatment (Fig. 1). Riluzole (110 M) displayed signif-
icant protective effects against L-DOPA toxicity with
respect to both morphology and cell count of THir cells FIG. 3. Effects of riluzole on toxicity induced by MPP on DA
in mesencephalic cultures (Figs. 1 and 7A). neurons after 48 h. A: THir neurons in mesencephalic cultures. In
SH-SY5Y cells showed only a mild susceptibility to all experiments the cells were cultured for 4 days before treat-
L-DOPA, with a loss of cell viability (measured with the ment with various concentrations of MPP alone () or in com-
MTT assay) to 65% of control after incubation with 300 bination with 1 M riluzole () for 48 h. Survival of THir neurons
is expressed as a percentage of the untreated control. The
M for 24 h. However, this effect was completely control value was 466 123 THir cells per well. B: Human
blocked by 10 M riluzole (data not shown). Therefore, dopaminergic neuroblastoma SH-SY5Y cells. The cells were
we used 6-OHDA as another dopaminergic toxin acting incubated with MPP alone () or in combination with 1 () or 10
especially via extracellular formation of free radicals and M () riluzole for 48 h. Cell viability was assessed by the MTT
method and presented as a percentage of untreated controls.
quinones (Tiffany-Castiglioni et al., 1982; Michel and Data are mean SEM (bars) values of at least three independent
Hefti, 1990; Abad et al., 1995; Storch et al., 2000c) to experiments. *p 0.05, **p 0.01 compared with untreated
investigate possible antioxidant effects of riluzole. In control; p 0.05 compared with MPP alone.

J. Neurochem., Vol. 75, No. 6, 2000

 PROTECTION OF DOPAMINE NEURONS BY RILUZOLE 2265

rons (Peluffo et al., 1997). Riluzole was found to induce

widespread neuronal as well as glial cell death in both
culture systems used at concentrations higher than those
needed for maximal protective effects (100 M for
both cell types). This is consistent with findings from
Koh et al. (1999) in primary cortical cultures from mice,
showing neuronal death at concentrations of 100 M
with classic features of apoptosis.
Riluzole promotes survival of motoneurons in vitro by
stimulating trophic activity produced by spinal astrocytes
(Peluffo et al., 1997), suggesting an indirect action of
riluzole on neuronal survival. However, in DA neurons
this is unlikely because addition of striatal medium con-

FIG. 4. Effects of conditioned medium (CM) collected from stri-

atal cultures incubated with various concentrations of riluzole for
36 h on MPP-induced toxicity on THir cells in recipient mes-
encephalic cultures after 4 days in vitro. CM was collected 24 h
after termination of riluzole treatment, and mesencephalic cul-
tures were incubated for 48 h with CM (1:2 diluted with defined
medium) alone or in combination with 3 M MPP. Unpaired t
test revealed no statistically significant difference in THir cell
count in cultures treated with CM (conditioned with various
concentrations of riluzole) and MPP relative to cultures treated
with MPP alone. Survival of THir neurons is expressed as a
percentage of the untreated control (no CM). The control value
was 356 25 THir cells per well. Data are mean SEM (bars)
values of at least three independent experiments.

peroxides in mesencephalic cultures (Table 1) were in

the same range as that reported in mouse cortical cultures
(Koh et al., 1999). Riluzole displayed a mild but nonsig-
nificant decrease of this basal lipid peroxidation level
(Table 1). We used iron-III as an established generator of
membrane lipid peroxidation in vitro (Koh et al., 1999),
and indeed a 24-h exposure to 100 M Fe(III)Cl3 sig-
nificantly increased lipid peroxidation in mesencephalic
cultures to 217 3% of control (Table 1). Furthermore,
L-DOPA also caused increased lipid peroxidation levels
in this culture system. As shown in Table 1, riluzole
significantly reduced lipid peroxidation in primary mes-
encephalic cultures induced by exposure to both Fe(II-
I)Cl3 (100 M) and L-DOPA (100 M) for 24 h.

DISCUSSION
The study presented here shows that riluzole exerts
dose-dependent protective effects on DA neurons in
mesencephalic cultures against toxic injuries caused by
the selective dopaminergic toxins MPP, L-DOPA, and
6-OHDA. The protective effect of riluzole toward dopa-
minergic cells (110 and 1100 M for primary DA cells
and dopaminergic neuroblastoma SH-SY5Y cells, re-
spectively) was in the same dose range used for the
induction of similar effects on cortical or motoneuron
cultures (Estevez et al., 1995; Mary et al., 1995; Koh
et al., 1999). However, the protective effect of riluzole
toward dopaminergic cells was independent of trophic
effects, in contrast to its effect on rat cultured motoneu-
FIG. 5. Effects of riluzole on changes of indices of cellular en-
ergy metabolism caused by MPP on human SH-SY5Y neuro-
blastoma cells. A: Intracellular ATP content. The cells were
treated with MPP (30, 100, or 300 M) alone or in combination
with riluzole (10 or 100 M) for 48 h. Then the intracellular net
ATP content was analyzed in perchloric acid cell extracts using
an HPLC method. Control value for intracellular ATP was 1.33
0.25 nmol per 106 cells. B: Intracellular net NADH content. The
cells were treated with 100 M MPP alone or in combination
with 30 M riluzole for 12 h. Then the intracellular net NADH
content was analyzed in alkaline cell extracts. The control value
for intracellular NADH was 1.12 0.12 nmol/mg protein. Data
are mean SEM (bars) values of at least three independent
experiments. *p 0.05, **p 0.01 compared with untreated
control; p 0.05, p 0.01 compared with MPP alone.

J. Neurochem., Vol. 75, No. 6, 2000

2266 A. STORCH ET AL.

lar maintenance of the Na gradient across the cell

membrane, needs a large part of cellular energy in nerve
cells [up to 70% of cellular energy is consumed by
Na,K-ATPase (Pedersen and Carafoli, 1987; Urenjak
and Obrenovitch, 1998)], reduction of energy demand by
blocking multiple ion channels (Benoit and Escande,
1991; Debono et al., 1993; Herbert et al., 1994; Hubert
et al., 1994; Bausch and Roy, 1996; Song et al., 1997)
may be responsible for the effect of riluzole on cellular
energy metabolism. However, our data do not fully rule
out that a partial recovery of ATP synthesis by riluzole

FIG. 6. Effects of riluzole on DA and MPP uptake in mesence-

phalic cultures. Cells were cultured for 3 days before uptake
experiments. Then cultures were pretreated with 10 M riluzole
for 30 min, before [3H]DA and [3H]MPP uptake was measured
in sister cultures in the presence and absence (control) of 10 M
riluzole. Control values were 1.4 0.35 and 1.2 0.19 pmol/min
per dish for specific DA and MPP uptake, respectively. Specific
uptake accounted for 85% of total uptake of both substances.
A t test revealed no statistically significant difference in uptake of
DA or MPP in the presence of riluzole relative to controls
(absence of riluzole). Data are mean SEM (bars) values of three
independent experiments.

ditioned with riluzole did not show any effect on THir

cell survival or MPP-induced toxicity in mesencephalic
cultures. The potential of riluzole in protecting DA neu-
rons against toxins acting in a few hours in vitro, such as
6-OHDA and L-DOPA (Michel and Hefti, 1990; Storch
et al., 2000a,c), supports the suggestion that riluzole acts
directly on DA neurons because production of neurotro-
phic activity by glial cells induced by mechanical or
toxic injuries is a slow process and requires several hours
to days (Nieto-Sampedro et al., 1982; Rudge et al.,
1995).
The mechanism by which riluzole exerts its neuropro-
tective effect on DA neurons in culture appears to in-
clude multiple and independent ways of action: (a) Ri-
luzole normalizes cellular energy supply altered by the
complex I inhibitor MPP, and (b) riluzole decreases
lipid peroxidation caused by iron-III and L-DOPA in
mesencephalic cultures. Riluzole decreases the intracel-
lular ATP depletion caused by MPP in dopaminergic
neuroblastoma SH-SY5Y cells. It is most likely that this
FIG. 7. Effects of riluzole on survival of DA neurons exposed to
normalization of cellular energy supply is responsible for various concentrations of L-DOPA and 6-OHDA. A: THir neurons
the protective effect because ATP depletion in this cell in mesencephalic cultures. In all experiments the cells were
system is the main cause of cell death induced by MPP cultured for 4 days before treatment with various concentrations
(Storch et al., 2000b,c). This suggestion is supported by of L-DOPA alone or in combination with riluzole (110 M) for
24 h. Survival of THir neurons is expressed as a percentage of
the comparable potencies of riluzole in normalizing ATP the untreated control. The control value was 581 111 THir cells
levels and reducing toxicity. However, riluzole does not per well. B: Human dopaminergic neuroblastoma SH-SY5Y
alter the intracellular increase of NADH level caused by cells. The cells were incubated with 6-OHDA alone or in combi-
MPP, suggesting that inhibition of NADH oxidation by nation with riluzole (1100 M) for 24 h. Cell viability was as-
mitochondrial complex I by MPP is not altered by sessed by the MTT method and presented as a percent of
untreated controls. Data are mean SEM (bars) values of at
riluzole. Therefore, riluzole seems to act indirectly on least three independent experiments. *p 0.05, **p 0.01
cellular energy metabolism, most likely by reducing cel- compared with untreated control; p 0.05, p 0.01 com-
lular ATP consumption. As ion homeostasis, in particu- pared with L-DOPA alone (A) or 6-OHDA alone (B).

J. Neurochem., Vol. 75, No. 6, 2000

 PROTECTION OF DOPAMINE NEURONS BY RILUZOLE
TABLE 1. Riluzole blocks lipid peroxidation in
mesencephalic cultures induced by Fe3 and L-DOPA
Condition TBARS (nmol/mg of protein)
Control 1.572 0.064
Riluzole (10 M) 1.315 0.090 (84 7%)
Fe3 (100 M) 3.405 0.091 (217 3%)a
Riluzole (10 M) 2.169 0.227 (138 10%)b
L-DOPA (100 M) 2.547 0.200 (162 8%)a
Riluzole (10 M) 1.775 0.085 (113 7%)b
Mesencephalic cultures were exposed after 4 days in vitro to the
various conditions given for a 24-h interval. TBARS were quantified as
described in Materials and Methods. Data are mean SE values of
three independent experiments. Numbers in parentheses are percent-
ages of the control.
a
p 0.01 compared with untreated control.
b
p 0.05 compared with Fe(III)Cl3 or L-DOPA alone.
may be another factor involved in the protective mech-
anism of riluzole. We believe that the effects of riluzole
on cellular energy supply most likely account for neuro-
protection of riluzole in animal models of PD using
MPTP as the parkinson-inducing agent (Benazzouz
et al., 1995; Bezard et al., 1998; Urenjak and Obreno-
vitch, 1998; Boireau et al., 2000).
The second way of action of riluzole is its direct
antioxidative effect: The present study shows that ri-
luzole reduces lipid peroxidation induced by Fe3 and
L-DOPA in primary mesencephalic cultures. These data
are consistent with findings from Koh et al. (1999) show-
ing reduced lipid peroxidation and inhibition of cytosolic
phospholipase A2, an enzyme that is linked to oxidative
stress (Janssen-Timmen et al., 1994). Consistent with
these data, riluzole attenuates oxidative injuries on cul-
tured DA neurons (present study), in primary cortical
cultures (Koh et al., 1999), and in animal models of PD
produced with 6-OHDA (Barneoud et al., 1996), where
oxidative stress may play a pivotal role (for review, see
Cadet and Brannock, 1998). It is interesting that riluzole
shows protective effects against in vitro toxicity of L-
DOPA, a substance that is widely used in the treatment
of parkinsonian patients and that may have toxic effects
on DA neurons also in vivo (Blunt et al., 1993; for
review, see Ling et al., 1996).
As both MPP (Gerlach et al., 1991; Przedborski and
Jackson-Lewis, 1998; Storch et al., 1999) and probably
6-OHDA (Cadet and Brannock, 1998; Storch et al.,
2000c) enter the DA neuron via the DAT molecule to
exert their toxicity, a possible mechanism by which
riluzole protects DA neurons is the blockade of the
uptake of these compounds by the DAT. The data pre-
sented from uptake studies using DA and MPP dem-
onstrate that inhibition of the DAT is not the mechanism
responsible for protection against dopaminergic toxicity
of these substances. This is consistent with our finding
that riluzole does not prevent inhibition of NADH oxi-
dation by MPP (see above), suggesting that cellular and
mitochondrial uptake of MPP is not altered by riluzole.
2267
Our data do not rule out that direct antiexcitotoxic
effects of riluzole, i.e., inhibition of glutamatergic neu-
rotransmission (Beal, 1992; Estevez et al., 1995; Mary
et al., 1995; Doble, 1999), are involved in the protection
of riluzole on DA neurons in our cell system, but the
strong correlation between effects on cellular energy
supply/oxidative stress and cytoprotection suggests that
direct antiexcitotoxic properties of riluzole do not play a
major role in our experiments. Furthermore, the NMDA
receptor antagonist MK-801 does not protect against
dopaminergic toxicity of MPP in primary mesence-
phalic cultures (Michel and Agid, 1992), and, in addi-
tion, -amino-3-hydroxy-5-methylisoxazole-4-propionate/
kainate antagonists failed to protect DA neurons against
toxicity induced by MPTP/MPP (Klockgether et al., 1991;
Turski et al., 1991). These data suggest that direct excito-
toxicity only plays a minor role in MPP toxicity toward
dopaminergic neurons in vitro.
We conclude that treatment with riluzole leads to a
protection of DA neurons in vitro against neuronal inju-
ries by (a) impairment of cellular energy metabolism and
(b) oxidative stress. With deficiency in cellular energy
metabolism and/or oxidative injury as the most likely
candidate mechanisms for neurodegeneration in PD
(Dunnett and Bjorklund, 1999), the present results sug-
gest that riluzole is a potential candidate as neuroprotec-
tive agent in PD as it can independently protect mesen-
cephalic DA neurons against both types of injury. Fur-
thermore, riluzole is protective against toxic effects of
L-DOPA in vitro, which is used in the treatment of PD.
Current clinical studies have to prove whether the effects
of riluzole can slow down the progression of PD.
Acknowledgment: The authors would like to thank Sinje
Jankowski for excellent technical assistance, Heike Blessing
and Vera Lehmensiek for help in preparations and treatments of
mesencephalic cultures, and Dr. Wilhelm Fischer for providing
riluzole and helpful comments during the study. This study was
supported in part by the Rhone Poulenc-Rorer Company, Ger-
many.
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