β-N-methylamino-L-alanine induces oxidative stress and glutamate release through
action on system Xc−
XiaoQian Liu, Travis Rush, Jasmine Zapata, Doug Lobner⁎
Department of Biomedical Sciences, Marquette University, 561 N. 15th Street, Rm 446 Milwaukee, WI 53233, USA
a b s t r a c t a r t i c l ei n f o
Received 6 January 2009
Revised 12 March 2009
Accepted 7 April 2009
Available online 15 April 2009
β-N-methylamino-L-alanine (BMAA) is a non-protein amino acid implicated in the neurodegenerative
disease amyotrophic lateral sclerosis/Parkinson–dementia complex (ALS/PDC) on Guam. BMAA has
recently been discovered in the brains of Alzheimer's patients in Canada and is produced by various
species of cyanobacteria around the world. These findings suggest the possibility that BMAA may be of
concern not only for specific groups of Pacific Islanders, but for a much larger population. Previous studies
have indicated that BMAA can act as an excitotoxin by acting on the NMDA receptor. We have shown that
the mechanism of neurotoxicity is actually three-fold; it involves not only direct action on the NMDA
receptor, but also activation of metabotropic glutamate receptor 5 (mGluR5) and induction of oxidative
stress. We now explore the mechanism by which BMAA activates the mGluR5 receptor and induces
oxidative stress. We found that BMAA inhibits the cystine/glutamate antiporter (system Xc−) mediated
cystine uptake, which in turn leads to glutathione depletion and increased oxidative stress. BMAA also
appears to drive glutamate release via system Xc−and this glutamate induces toxicity through activation of
the mGluR5 receptor. Therefore, the oxidative stress and mGluR5 activation induced by BMAA are both
mediated through action at system Xc−. The multiple mechanisms of BMAA toxicity, particularly the
depletion of glutathione and enhanced oxidative stress, may account for its ability to induce complex
© 2009 Elsevier Inc. All rights reserved.
In the 1959's it was observed that a substantial number of
Chamorros, the native people of Guam, began developing a disease
which showed combined symptoms of amyotrophic lateral sclerosis
and Parkinson's dementia complex (ALS/PDC). The idea that β-N-
methylamino-L-alanine (BMAA) may be involved in this disease began
in the 1989's when it was found that BMAAwas present in cycad seeds
which were consumed by the Chamorros (Nunn et al.,1987) and that
injection of BMAA into monkey brains induced a Parkinson-like
disease (Spencer et al., 1987). Since its initial proposal, the BMAA
hypothesis for the development of ALS/PDC on Guam has been
controversial (Cox and Sacks, 2002; Papapetropoulos, 2007). It was
challenged by findings that the levels of BMAA in cycad seeds are too
low to cause damage to the brain or the spinal cord, particularly
because the Chamorros thoroughly wash the cycad seeds, leading to
very low levels of BMAA being consumed (Duncan et al., 1990). The
BMAA hypothesis was largely abandoned until the last few years.
A number of recent studies have brought the BMAA hypothesis
back into prominence. First, it was shown that BMAA is bio-
magnified. BMAA is produced by cyanobacteria that live on cycad
plants; it accumulates in the cycad seeds, which are eaten by fruit
bats, which are in turn eaten by the Chamorros (Cox et al., 2003).
Second, it was shown that BMAA can become protein-associated.
This property allows for BMAA to build up in tissue and provides a
mechanism for slow release (Murch et al., 2004a). This slow release
may provide a possible explanation for the delayed onset of ALS/
PDC following the time of BMAA consumption (Ince and Codd,
2005). Third, cyanobacteria present throughout the world have
been shown to produce BMAA (Cox et al., 2005; Banack et al.,
2007; Esterhuizen and Downing, 2008; Johnson et al., 2008;
Metcalf et al., 2008). Also, BMAA was found not only in brain
samples of ALS/PDC patients from Guam, but also in the brains of
Alzheimer's disease patients from Canada, but not in patients who
died of other causes (Murch et al., 2004b). These results suggest
that BMAA may be of concern not only for people on select Pacific
islands, but for a much larger population. Fourth, BMAA at lower
concentrations than previously believed are neurotoxic. The original
studies in cortical cell culture found that very high BMAA concen-
trations (1–3 mM) were required to induce neuronal death (Ross
et al., 1987; Weiss and Choi, 1988; Weiss et al., 1989). A more recent
study found that BMAA concentrations as low as 30 μM can cause
selective death of motor neurons (Rao et al., 2006), and we found that
BMAA concentrations as low as 10 μM can enhance neuronal death
Experimental Neurology 217 (2009) 429–433
⁎ Corresponding author. Fax: +1 414 288 6564.
E-mail address: Doug.Lobner@marquette.edu (D. Lobner).
0014-4886/$ – see front matter © 2009 Elsevier Inc. All rights reserved.
Contents lists available at ScienceDirect
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induced by amyloid-β or 1-methyl-4-phenylpyridinium ion (MPP+)
(Lobner et al., 2007).
Given the potential relevance of BMAA consumption to neurode-
generative diseases it is important to determine the mechanism of
BMAA induced neuronal death. We have previously shown that
BMAA induces neuronal death through 3 distinct mechanisms, acti-
vation of NMDA and mGluR5 receptors, and induction of oxidative
stress (Lobner et al., 2007). Through electrophysiological recording it
has been shown that BMAA directly acts on the NMDA receptor (Ross
et al., 1987; Weiss and Choi, 1988; Brownson et al., 2002; Lobner
et al., 2007). The current studies were designed to determine how
BMAA activates mGluR5 receptors and induces oxidative stress.
Evidence is presented that the cystine/glutamate antiporter (system
Xc−) plays an important role in these effects. System Xc−involves
the transport of cystine into the cell in exchange for glutamate being
transported out of the cell. Given the functions of system Xc−it
seems likely that it plays an important role in neuronal survival and
death. By releasing glutamate it can increase extracellular glutamate
levels and potentially cause excitotoxicity. Through providing cystine
uptake it can regulate cellular glutathione levels and in this way
determine whether oxidative stress induced neuronal death will
occur. We find that the effects of BMAA on neuronal death involve
actions on system Xc−to both inhibit cystine uptake and increase
Materials and methods
Timed pregnant Swiss Webster mice were obtained from Charles
River Laboratories (Wilmington, DE, USA).
PerkinElmer Life and Analytical Sciences (Boston, MA). 5-(and -6)-
carboxy-2′7′-dichlorodihydrofluorescein diacetate (carboxy-
H2DCFDA) was from Molecular Probes (Eugene, OR). All other
chemicals were obtained from Sigma (St. Louis, MO).
35S-cystine was from
Cortical cell cultures
Mixed cortical cell cultures containing neuronal cells were pre-
pared from fetal (15–16 day gestation) mice as previously described
(Lobner, 2000). Dissociated cortical cells were plated on 24 well
plates coated with poly-D-lysine and laminin in Eagles' Minimal
Essential Medium (MEM, Earle's salts, supplied glutamine-free)
supplemented with 5% heat-inactivated horse serum, 5% fetal bovine
serum, 2 mM glutamine and glucose (total 21 mM). Cultures were
maintained in humidified 5% CO2incubators at 37 °C. Mice were
handled in accordance with a protocol approved by our Institutional
Animal Care Committee.
Induction of neuronal death
All experiments were performed on mixed cultures 13–15 days in
vitro (DIV). Toxicity was induced by exposure to the toxic agents for
24 h in media as described for plating except without serum. All
exposure media contained 26 mM NaHCO3, as it has been shown
previously that HCO3
mediated BMAA toxicity (Weiss and Choi, 1988).
−is required for expression of NMDA receptor
Assay of neuronal death (LDH release)
Cell death was assessed in mixed cultures by the measurement of
lactate dehydrogenase (LDH), released from damaged or destroyed
cells, in the extracellular fluid 24 h after the beginning of the insult.
Blank LDH levels were subtracted from insult LDH values and results
normalizedto100% neuronal death caused by500 mMNMDA. Control
experiments have shown previously that the efflux of LDH occurring
from either necrotic or apoptotic cells is proportional to the number of
cells damaged or destroyed (Koh and Choi, 1987; Lobner, 2000). Glial
cell death (assessed by trypan blue staining) was not observed in any
of the current studies. Therefore results are presented as percent
Uptake of cystine was measure by exposure of cultures to
35S-cystine (2 μCi/ml) for 20 min in the presence or absence of
3 mM BMAA and/or 1 mM S-4-carboxyphenyl glycine (CPG).
Following the exposure to
3 times and dissolved in 1% SDS (250 μl). An aliquot (200 μl)
was removed and added to scintillation fluid for counting. Values
were normalized to control
35S-cystine, the cultures were washed
35S-cystine uptake (20 min exposure
35S-cystine without BMAA or CPG).
Total glutathione was assayed using a modification of a previous
method (Baker et al., 1990; Lobner et al., 2003). Briefly, following
exposure to BMAA for 3 h, cells were washed with a HEPES buffered
saline solution, dissolved in 200 μl of 1% sulfosalicylic acid, and
centrifuged. A 25 μl aliquot of the supernatant was combined with
150 μl of 0.1 M phosphate/5 mM EDTA buffer, 10 μl of 20 mM
dithiobis-2-nitrobenzoic acid, 100 μl of 5 mM NADPH, and 0.2 U of
glutathione reductase. Total glutathione was determined by kinetic
analysis of absorbance changes at 402 nm for 1.5 min, with con-
centrations determined by comparison to a standard curve.
Assay of intracellular oxidative stress
Oxidative stress was assayed by measuring dichlorofluorescein
oxidation using a fluorescent plate reader following a modification
of a previous method (Wang and Joseph, 1999; Lobner et al., 2007).
Cultures were exposed to 3 mM BMAA for 3 h in the presence of 5-
(and -6)-carboxy-2′7′-dichlorodihydrofluorescein diacetate (car-
boxy-H2DCFDA) (10 μM). The carboxy-H2DCFDA is de-esterified
within cells to form a free acid that can then be oxidized to the
fluorescent 2′7′-dichlorofluorescein (DCF). After the exposure to
carboxy-H2DCFDA, cultures were washed three times with culture
media lacking serum. Fluorescence was then measured using a
Fluoroskan Ascent fluorescence plate reader (ThermoLabsystems).
The excitation filter was set at 485 nm and emission filter at
538 nm. Background fluorescence (no carboxy-H2DCFDA added)
was subtracted and the results normalized to control conditions
(carboxy-H2DCFDA added, but no BMAA exposure).
Analysis of glutamate release
Glutamate release was measured following exposure to 3 mM
BMAA or cystine for 1 h. Experiments were performed in the presence
of100 μMDL-threo-β-benzyloxyaspartate (TBOA) toblockreuptakeof
released glutamate by Na-dependent glutamate transporters and in
the presence of 10 μM MK-801 to block potential injury induced
glutamate release. Samples of the bathing media fromthe cell cultures
were assayed for glutamate by using phenylisothiocyanate (PITC)
derivatization, HPLC (Agilent 1100) separation using a Hypersil-ODS
reverse phase column, and ultraviolet detection at a wavelength of
254 nm (Cohen et al., 1986; Lobner and Choi, 1996). 200 μl of the
bathing media is derivatized with 100 μl of PITC, methanol,
triethylamine (2,7,4) and dried under vacuum. These samples are
then reconstituted in solvent consisting of 0.14 M sodium acetate,
0.05% TEA, 6% acetonitrile and brought to pH 6.4 with glacial acetic
acid. The above solvent is used as the mobile phase with the column
being washed between each sample run in 60% acetonitrile, 40%
X. Liu et al. / Experimental Neurology 217 (2009) 429–433
water. Media glutamate concentrations were calculated by norma-
lizing to glutamate standards. Glutamate measurements were found
to be linear over the range 0.1–10 μM.
Differences between test groups were examined for statistical
significance by means of one-way ANOVA followed by the Bonferroni
t-test, with pb0.05 being considered significant.
BMAA toxicity involves the NMDA receptor, mGLuR5 receptor, and
As has been shown before we found that blockade of NMDA
receptors with MK-801 provided significant protection against high
concentration BMAA toxicity (Ross et al., 1987; Weiss et al., 1989)
(Fig. 1). However, as in the previous studies, the protection was not
complete, suggesting additional mechanisms of BMAA toxicity. As we
have shown previously (Lobner et al., 2007) the mGluR5 antagonist
6-methyl-2-[phenylethynyl]-pyridine (MPEP), and the free radical
scavenger, trolox, were not protective by themselves, but provided
additional protection against BMAA toxicity beyond that provided by
MK-801 (Fig. 1). Furthermore, the combination of these agents pro-
vided the greatest protection, suggesting that they were acting
through distinct mechanisms. The MPEP and trolox were not pro-
tective against BMAA toxicity without MK-801 present likely because
of the overwhelming toxic effects of BMAA on the NMDA receptor in
the absence of MK-801. Activation of AMPA/kainite receptors does
not appear to be involved as we have shown previously that blocking
AMPA/kainite receptors with CNQX is not protective even in the
presence of MK-801 (Lobner et al., 2007). A high concentration of
BMAA (3 mM) was used so that complete neuronal death was
induced and the different mechanisms of toxicity could be studied.
BMAA induces oxidative stress through inhibition of system
Xc−mediated cystine uptake
One possibility for induction of oxidative stress by BMAA is that
it inhibits cystine uptake. Cystine is the precursor for production of
the endogenous free radical scavenger, glutathione. We found that
BMAA did in fact greatly attenuate cystine uptake (Fig. 2). Also, the
cystine uptake was largely blocked by the cystine/glutamate anti-
porter (system Xc−) inhibitor (S)-4-carboxyphenylglycine (CPG),
and BMAA did not cause inhibition of uptake in the presence of CPG
(Fig. 2). Therefore, it is likely that BMAA attenuates the uptake of
cystine by inhibition of system Xc−.
Since BMAA inhibits cystine uptake it would be expected to
decrease cellular glutathione levels. After a 3 hour treatment with
BMAA, glutathione levels were decreased by about 50% (Fig. 3).
Three hour exposure was chosen because at this time point there was
no significant neuronal death observed (less than 10% by the LDH
Since BMAA causes a decrease in the levels of the endogenous
free radical scavenger glutathione, the total oxidative stress of the
cells should be increased. We measured cellular oxidative stress with
the fluorescent dye dichlorofluorescein (DCF). Treatment with BMAA
for 3 h caused a significant increase in oxidative stress which was
blocked by the free radical scavenger trolox (Fig. 4).
Fig.1. BMAA inducedtoxicityoccurs throughmultiple mechanisms. MK:10μMMK-801;
MPEP: 50 μM 6-methyl-2-[phenylethynyl]-pyridine (MPEP); trolox: 100 μM trolox. Bars
show % neuronal cell death (mean±s.e.m., n=16–20) quantified by measuring release
of LDH, 24 h after the beginning of the insult. ⁎ indicates significant difference.
Fig. 2. BMAA inhibits system Xc−mediated cystine uptake. BMAA: 3 mM BMAA;
CPG: 1 mM S-4-carboxyphenyl glycine (CPG). Bars show35S-cystine uptake during a
20 minute exposure presented as % control (mean±s.e.m., n=16). ⁎ indicates
significant difference from control.
Fig. 3. BMAA decreases cellular glutathione levels. BMAA: 3 mM BMAA. Bars show
glutathione levels following a 3 hour BMAA exposure presented as % control
(mean±s.e.m., n=16). ⁎ indicates significant difference from control.
Fig. 4. BMAA induces oxidative stress. BMAA: 3 mM BMAA; trolox: 100 μM trolox.
5-(and -6)-carboxy-2′7′-dichlorodihydrofluorescein diacetate (10 mM) was added to
the cultures during a 3 hour exposure to BMAA. Bars show % control fluorescence
(mean±s.e.m., n=12). ⁎ indicates significant difference from control.
X. Liu et al. / Experimental Neurology 217 (2009) 429–433
BMAA and cystine induce glutamate release and excitotoxicity
The inhibition by BMAA of system Xc−mediated cystine uptake
suggests that BMAA competes with cystine; if like cystine, it is trans-
ported, the high levels of BMAA used in these studies should lead to
increased glutamate release. We did in fact find that 3 mM BMAA
increased extracellular glutamate. A similar increase was also caused
by 3 mM cystine (Fig. 5).
Since BMAA appears to induce glutamate release via system Xc−,
it is possible that this glutamate is responsible for at least part of the
glutamate receptor mediated neuronal death. Because 3 mM cystine
caused an increase in extracellular glutamate, it may also induce
glutamate receptor mediated neuronal death. We found that 3 mM
cystine did induce neuronal death (Fig. 6). The death was less than
that caused by 3 mM BMAA, likely due to the fact that cystine is not
a direct NMDA receptor agonist like BMAA. However, the toxicity
was partially blocked by either MPEP or MK-801. No protection was
afforded by trolox, as would be expected since high concentration of
cystine should not induce oxidative stress.
We found that BMAA inhibits system Xc−mediated cystine uptake
leading to decreased cellular glutathione. The process of glutathione
production in neurons involves a complex series of steps in which
cystine is taken up primarily into astrocytes (Sagara et al., 1993). The
glutathione produced by astrocytes is released and converted
extracellularly into cysteine, which is taken up by neurons and used
by them to produce glutathione (Wang and Cynader, 2000). Whether
the action of BMAA to inhibit cystine uptake was primarily on neurons
orastrocytes was not determined. Therefore it is not known if BMAA is
initially acting to inhibit cystine uptake into neurons, or if it inhibits
the uptake of cystine into astrocytes and prevents their release of
glutathione and therefore restricts the supply of cysteine to neurons.
The data presented here strongly suggests that BMAA not only
inhibits cystine uptake, but also drives system Xc−to release glu-
tamate and that this glutamate induces neuronal death by acting on
mGluR5 receptors, and possibly NMDA receptors. The evidence for
this is that BMAA and cystine at the same concentration (3 mM)
increase extracellular glutamate and they both induce mGluR5
mediated neuronal death. There is no evidence that either of these
compounds directly activates mGluR5. Unfortunately, CPG could not
be used to block the glutamate release because while it is a fairly
poor substrate for system Xc−(Patel et al., 2004), at the high
concentrations needed to compete with the high concentrations of
BMAA and cystine used in these studies it stimulated glutamate
release by itself (data not shown). Why in our studies there was
some protection against cystine induced toxicity by MK-801 is not
clear. There is no evidence that cystine is a direct NMDA receptor
agonist. However, cystine may be converted into cysteine, which is
an NMDA receptor agonist (Pullan et al., 1987). Alternatively, the
glutamate release stimulated by cystine via system Xc−may act to
stimulate NMDA receptors.
There is growing evidence that system Xc−is involved in neuronal
death. It has been known for many years that inhibition of cystine
uptake, probably mediated by system Xc−, can induce neuronal death
(Murphy et al., 1989, 1990), and there is recent evidence that
glutamate release from system Xc−can be neurotoxic. It has been
shown that activated astrocytes release glutamate via systemXc−that
can kill cortical neurons (Fogal et al., 2007). Also, microglia can release
glutamate via system Xc−that can kill cerebellar granule cells (Piani
and Fontana, 1994), oligodendrocytes (Domercq et al., 2007), and
enhance amyloid-β induced neuronal death in cortical cultures (Qin
et al., 2006). The system Xc−mediated glutamate release from
microglia appears to be induced by oxidative stress (Barger et al.,
2007), setting up a potential feedback loop between microglia and
neurons. Finally, glutamate release fromdendritic cells via systemXc−
can inhibit T cell activation through actions on mGluR5 receptors
(Pacheco et al., 2006). Interestingly, another amino acid implicated in
neurological diseases, beta-N-oxalyl-L-alpha,beta-diaminopropionic
acid (ODAP), is also transported by system Xc−(Chase et al., 2007).
In the current study we find evidence that oxidative stress induced by
the inhibition of cystine uptake and excitotoxicity induced by system
Xc−mediated glutamate release both play a role in BMAA induced
The finding that BMAA not only inhibited cystine uptake by system
Xc−, but also stimulated glutamate release, suggests that it was
transported by system Xc−. This transport provides a mechanism by
which BMAA can accumulate in cells. There is evidence that BMAA
may be incorporated into proteins (Murch et al., 2004a) and could
therefore potentially play a role in the protein misfolding found in
neurodegenerative diseases (Uversky, 2008).
The importance of BMAA acting on system Xc−to induce oxidative
stress and excitotoxicity is that this may partially account for the
different types of neurological diseases that have been associated with
BMAA consumption. It is possible that the different actions of BMAA,
in association with varying underlying conditions, may lead to the
multiple disorders. Most neurodegenerative diseases appear to
involve NMDA receptor mediated excitotoxicity (Lipton, 2004) and
oxidative stress (Cui et al., 2004), and mGluR5 receptor activation has
been implicated in Parkinson's disease (Marino et al., 2003). All of
these mechanisms of neuronal injury may be enhanced by BMAA. The
finding that BMAA causes a depletion of cellular glutathione is of
particular importance. There is evidence that glutathione depletion
plays a role in Alzheimer's disease, Parkinson's disease, and ALS (Bains
and Shaw,1997; Liu et al., 2004; Zeevalk et al., 2008). The type of low
level, long-term, depletion of glutathione that may be caused by the
Fig. 5. BMAA and cystine stimulate glutamate release. BMAA: 3 mM BMAA; cystine:
3 mM cystine. Bars show extracellular glutamate levels following a 1 hour BMAA or
cystine exposure (mean±s.e.m., n=8). ⁎ indicates significant difference from control.
Fig. 6. Cystine-induced neuronal death is attenuated by MK-801 and MPEP. MK: 10 μM
MK-801, MPEP: 50 μM MPEP; trolox: 100 μM trolox. Bars show % neuronal cell death
(mean±s.e.m., n=24) quantified by measuring release of LDH, 24 h after the
beginning of the insult. ⁎ indicates significant difference from control (cystine alone).
X. Liu et al. / Experimental Neurology 217 (2009) 429–433
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