The role of excitotoxicity in the pathogenesis of amyotrophic lateral sclerosis
L. Van Den Bosch⁎, P. Van Damme, E. Bogaert, W. Robberecht
Neurobiology, Campus Gasthuisberg O&N2, PB1022, Herestraat 49, B-3000 Leuven, Belgium
Received 21 February 2006; received in revised form 4 May 2006; accepted 10 May 2006
Unfortunately and despite all efforts, amyotrophic lateral sclerosis (ALS) remains an incurable neurodegenerative disorder characterized by the
progressive and selective death of motor neurons. The cause of this process is mostly unknown, but evidence is available that excitotoxicity plays
an important role. In this review, we will give an overview of the arguments in favor of the involvement of excitotoxicity in ALS. The most
important one is that the only drug proven to slow the disease process in humans, riluzole, has anti-excitotoxic properties. Moreover, consumption
of excitotoxins can give rise to selective motor neuron death, indicating that motor neurons are extremely sensitive to excessive stimulation of
glutamate receptors. We will summarize the intrinsic properties of motor neurons that could render these cells particularly sensitive to
excitotoxicity. Most of these characteristics relate to the way motor neurons handle Ca2+, as they combine two exceptional characteristics: a low
Ca2+-buffering capacity and a high number of Ca2+-permeable AMPA receptors. These properties most likely are essential to perform their normal
function, but under pathological conditions they could become responsible for the selective death of motor neurons. In order to achieve this worst-
case scenario, additional factors/mechanisms could be required. In 1 to 2% of the ALS patients, mutations in the SOD1 gene could shift the
balance from normal motor neuron excitation to excitotoxicity by decreasing glutamate uptake in the surrounding astrocytes and/or by interfering
with mitochondrial function. We will discuss point by point these different pathogenic mechanisms that could give rise to classical and/or slow
excitotoxicity leading to selective motor neuron death.
© 2006 Elsevier B.V. All rights reserved.
Keywords: ALS; Neurodegeneration; Motor neuron; Ca2+metabolism; Glutamate; AMPA receptors; GluR2
1. What is excitotoxicity?
During glutamatergic neurotransmission, glutamate released
from the presynaptic neuron activates ionotropic glutamate
receptors present on the postsynaptic neuron (Fig. 1A).
Activation of these glutamate receptors results in the influx of
Na+and Ca2+ions into the cell, leading to depolarization and
ultimately to the generation of an action potential. Excitotoxi-
city is neuronal degeneration caused by over-stimulation of the
glutamate receptors. This concept was formulated in 1978 by
Olney  and was based on the observation that those amino
acids that induce neuronal death were the ones known to
activate excitatory amino acid receptors. Since then, experi-
mental evidence became available that excitotoxicity could
contribute to neuronal damage in stroke, neurotrauma, epilepsy,
and a number of neurodegenerative disorders including
amyotrophic lateral sclerosis (ALS) [2–4].
Rather artificially, two types of excitotoxicity are distin-
guished and denoted with the terms classical and slow
excitotoxicity . Classical excitotoxicity refers to neuronal
degeneration that occurs after an increase of the extracellular
glutamate concentration (Fig. 1B), while slow excitotoxicity is
the death of a weakened postsynaptic neuron in the presence of
normal synaptic glutamate levels (Fig. 1C).
In order to obtain classical excitotoxicity, an elevation of the
extracellular glutamate concentration to 2–5 μM is considered
sufficient to cause degeneration of neurons through excessive
stimulation of glutamate receptors [5,6]. Acute elevations of
glutamate are thought to induce neuronal damage in conditions
such as stroke, status epilepticus and neurotrauma. More
chronic and milder elevations of glutamate are believed to
underly excitotoxicity in neurodegenerative diseases. Elevated
Biochimica et Biophysica Acta xx (2006) xxx–xxx
BBADIS-62576; No. of pages: 15; 4C:
⁎Corresponding author. Tel.: +32 16 34 57 85; fax: +32 12 33 07 70.
E-mail address: Ludo.Vandenbosch@med.kuleuven.be
(L. Van Den Bosch).
0925-4439/$ - see front matter © 2006 Elsevier B.V. All rights reserved.
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extracellular glutamate concentrations can occur when the
release from presynaptic terminals is augmented or when the re-
uptake from the synaptic cleft is insufficient. This re-uptake is
assured by glutamate transporters present in neurons and
astrocytes. By far the most important glutamate transporter is
EAAT2/GLT-1 as it is widely expressed in astrocytes through-
out the central nervous system (CNS) and as it has the highest
affinity for glutamate. Elevated glutamate concentrations can
also arise if the intracellular glutamate content is released from
injured neurons. This release can result in excitotoxic death of
surrounding neurons and as a consequence could be involved in
the spread of the neurodegeneration.
In contrast to the classical excitotoxicity, no upregulation of
the synaptic glutamate concentration is needed to cause slow
excitotoxicity. In this case, normal glutamate receptor stimula-
tion of a weakened postsynaptic neuron is sufficient to damage
and kill the cell. Awell known example that could result in this
form of excitotoxicity is the disturbance of the mitochondrial
function resulting in an impaired energy state of the neuron.
This causes a drop in the intracellular ATP level. As a
consequence, the neuron cannot longer meet the demands of
its many ATP dependent processes, becomes damaged and
ultimately dies [7,8].
In both forms of excitotoxicity, the glutamatergic neuro-
transmission plays a pivotal role. Glutamate released from the
presynaptic neuron activates different types of glutamate
receptors present on the postsynaptic neuron. These glutamate
receptors are divided into ionotropic and metabotropic recep-
tors. The ionotropic glutamate receptors are ligand-gated cation
channels, while the metabotropic glutamate receptors are G-
protein coupled receptors that influence second messenger
systems. The ionotropic receptors can further be subdivided into
three classes according to their preferred synthetic agonist
[9,10]: AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole pro-
pionic acid), NMDA (N-methyl-D-aspartate) and KA (kainate)
receptors. Although, KA has a high affinity for KA receptors
resulting in rapidly desensitizing currents, it is mostly used as an
agonist of AMPA receptors since it also induces non-
desensitizing currents through these receptors. Functionally,
AMPA receptors are the most important glutamate receptors to
mediate fast excitatory transmission. On the other hand, NMDA
receptors mediate the late component of excitatory transmission
 and play a key role in the induction of synaptic plasticity
. The role of KA receptors in physiological and pathological
conditions is less clear.
At first, the NMDA receptor was considered to be uniquely
responsible for excitotoxicity . More recently, it became
apparent that the activation of AMPA receptors is at least as
important . The predominant mediator of neuronal injury is
Ca2+influx through NMDA receptors, Ca2+-permeable AMPA
receptors or voltage-gated Ca2+channels [15–17]. How Ca2+
entry can induce neuronal death is not yet completely
understood, but several possible mechanisms have been
reported. Excessive influx of Ca2+ions can result in the
activation of several enzymes, such as lipases, phospholipases,
proteases, endonucleases, protein phosphatases, protein kinase
C, xanthine oxidase and NO synthase. In addition, mitochon-
drial dysfunction due to increased Ca2+uptake in mitochondria
and subsequent formation of reactive oxygen species could also
contribute to excitotoxic cell death [18–20].
While NMDA receptors consist of NR1 and NR2(A–D)
subunits, AMPA receptors are tetramers composed of a variable
association of four subunits (GluR1–4) [21–25]. Both NMDA
and AMPA receptors are ligand-gated cation channels that
permit the influx of Na+and the efflux of K+. In addition, the
NMDA receptor is always Ca2+permeable, while he AMPA
Fig. 1. Glutamatergic neurotransmission and excitotoxicity. Under normal conditions,glutamate released from the presynaptic neuronactivates the NMDA and AMPA
receptors (R). This results in an influx of both Na+and Ca2+ions, the depolarization of the postsynaptic neuron and ultimately in an action potential. (B) Classical
excitotoxicityisinducedbyanelevationofthe extracellularglutamateconcentration.Thiscanbe causedbyan increasedreleaseof glutamateor adeficientre-uptakeof
glutamate into the astrocytes by the EAAT2/GLT-1 transporter. The excessive stimulation of the glutamate receptors gives rise to an increased intracellular
concentration of Na+and Ca2+ions and this can result in neuronal death. The disintegration of neuronal cells causes a further increase of extracellular glutamate and
amplifies the excitotoxic damage. (C) Slow excitotoxicity is characterized by the fact that the postsynaptic neuron becomes more sensitive to glutamate stimulation,
while the extracellular glutamate concentration is not increased. This sensitization of the neuron can be caused by changes in the properties of the glutamate receptors,
resulting in a higher increase of the intracellular Ca2+concentration. Alternatively, the vulnerability of neurons to normal glutamate stimulation can be increased due to
mitochondrial damage, resulting in energetically compromised neurons.
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receptor is permeable for Ca2+to a variable degree. The Ca2+
permeability of the AMPA receptor is determined by the
absence/presence of the GluR2 subunit in the receptor complex
(Fig. 2). Receptors containing GluR2 have a very low Ca2+
permeability compared to GluR2-lacking receptors . The
impermeability to Ca2+of GluR2-containing AMPA receptors
is due to the presence of a positively charged arginine at
position 586 (Q/R site) of the GluR2 peptide instead of the
genetically encoded neutral glutamine [27–29]. This arginine
residue at the Q/R site is introduced by the editing of GluR2 pre-
mRNA , which is virtually complete in most cell types.
Under pathological conditions, changes in subunit composition
can dramatically affect neuronal survival. The best studied
paradigm is the downregulation of GluR2 induced by ischemia,
which results in enhanced Ca2+influx and neuronal degener-
ation in vulnerable regions of the brain [31,32].
2. Excitotoxicity and ALS
In this review, we will focus on the role of excitotoxicity in
the pathology of ALS. This neurodegenerative disease is
characterized by the progressive and selective loss of motor
neurons in the motor cortex, the brainstem and the spinal cord.
This degeneration of motor neurons results in a progressive
paresis of bulbar, respiratory and limb muscles and leads to the
death of the patients usually 2 to 5 years after the diagnosis.
Familial ALS occurs in 5 to 10% of cases and predominantly
has an autosomal dominant inheritance. In 20% of familial
cases, mutations in the superoxide dismutase-1 (SOD1) gene on
chromosome 21q were identified . Mutations in SOD1 give
rise to a toxic ‘gain of function’ of the mutated protein.
Transgenic mice and rats that overexpress mutant SOD1
develop an age-dependent, selective motor neuron death
The most important argument for a role of excitotoxicity in
ALS is that riluzole, the only drug which proved effective
against disease progression in patients, has anti-excitotoxic
properties [36,37]. Riluzole slows the disease progression and
significantly increases survival by a few months. It was shown
that this drug inhibits the release of glutamate due to the
inactivation of voltage-dependent Na+channels on glutamater-
gic nerve terminals, as well as to activation of a G-protein-
dependent signal transduction process . Moreover, evidence
exists that riluzole can also block some of the postsynaptic
effects of glutamate by non-competitive inhibition of NMDA
and AMPA receptors [38,39]. In addition, postsynaptic effects
of riluzole on the γ-aminobutyric acid A (GABAA) receptor
have been described that could also contribute to its protective
We will start this part with an overview of the intrinsic
properties of motor neurons that could make these cells more
vulnerable to excitotoxicity. In the second and third part, we will
discuss the arguments in favor of the involvement of classical
and slow excitotoxicity in the selective motor neuron death
2.1. Selective vulnerability of motor neurons
Several lines of evidence indicate that motor neurons, both in
vitro and in vivo, are particularly vulnerable to AMPA receptor-
mediated excitotoxicity. Intrathecal or intraspinal administra-
tion of AMPA receptor agonists induced motor neuron
degeneration in animals, while NMDA failed to damage spinal
motor neurons [42–48]. Selective loss of motor neurons was
induced, especially when KA was used [43,48].
In organotypic rat spinal cord cultures (spinal cord slices
from postnatal rats, which can be kept in culture for several
weeks), motor neurons also proved to be particularly vulnerable
to AMPA receptor-mediated excitotoxicity [49,50]. Direct
application of AMPA receptor agonists resulted in selective
motor neuron loss, which could be prevented by antagonists of
Fig. 2. The Ca2+permeability of AMPA-type glutamate receptors is determined by the presence or absence of GluR2 subunit in the receptor complex. (A) In response
to glutamate (Glt) released from the presynaptic neuron, ionotropic AMPA receptors are activated. Most AMPA receptors only allow Na+to enter the cell, while a
special type of AMPA receptors is permeable for incoming Na+as well as Ca2+ions. (B) Detailed representation of a Ca2+-permeable (left) and a Ca2+-impermeable
AMPA receptor (right). If the receptor complex is composed of a combination of GluR1-3-4, it is permeable to Ca2+ions. If at least one GluR2 subunit is present in the
receptor complex,it is impermeableto Ca2+. This is dueto a positively charged arginine at position 586 that is only present in the GluR2 subunit if the GluR2mRNA is
edited. Under normal conditions this editing process that changes a CAG codon into a CGG codon is highly efficient and affects all GluR2 mRNAs.
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Similarly, it was demonstrated that motor neurons in culture
are susceptible to glutamate receptor agonists, especially those
stimulating AMPA receptors [16,51–55]. Using motor neurons
cultured on an astrocytic feeder layer, we showed that Ca2+
influx through Ca2+-permeable AMPA receptors was crucial for
inducing motor neuron death [17,56]. Other neurons were
resistant to AMPA receptor over-stimulation . Little or no
effect of agonists of NMDA receptors was apparent in these
cultured motor neurons , Moreover, depolarizing these
motor neurons by exposing them to a high extracellular K+
concentration was not sufficient to induce significant cell death
. As illustrated in Fig. 3, motor neurons seem to have a
number of intrinsic properties related to their Ca2+handling that
makes them particularly vulnerable to AMPA receptor mediated
excitotoxicity and that we will discuss below.
2.1.1. Low Ca2+-buffering capacity and role of mitochondria
The capacity of motor neurons to buffer increases in
intracellular Ca2+is limited due to the low expression level of
Ca2+-buffering proteins (Fig. 3). This low Ca2+-buffering
capacity could be essential under physiological conditions as
it allows rapid relaxation times of Ca2+transients in motor
neurons during high-frequency rhythmic activity . Howev-
er, this characteristic could make motor neurons more
susceptible to an excessive influx of Ca2+ions.
Immunostaining revealed that spinal motor neurons do not
express the Ca2+-binding proteins parvalbumin and calbindin
D28K, while less vulnerable motor neurons such as those in the
oculomotor, trochlear, abducens and Onuf's nucleus clearly
express Ca2+-binding proteins [59–61]. Similarly, parvalbumin
mRNA expression was high in ALS-resistant motor neuron
pools, while no measurable parvalbumin expression was found
in ALS-sensitive motor neurons . Electrophysiological
estimation of the Ca2+-buffering capacity in murine brainstem
and spinal cord slices confirmed a much higher Ca2+-buffering
capacity of oculomotor than hypoglossal or spinal motor
neurons [63–65]. Motor neurons from parvalbumin over-
expressing mice were shown to be more resistant to excitotoxic
stimuli. These cells showed a lower increase of intracellular
Ca2+and an increased survival after AMPA receptor
stimulation compared to normal motor neurons . Cross-
breeding of parvalbumin overexpressing mice with SOD1G93A
mice delayed disease onset with 17% and prolonged survival
by 11% .
A direct consequence of the lower amount of Ca2+-buffering
proteins in motor neurons is that mitochondria have to play a
more prominent role in Ca2+buffering in these cells (Fig. 3A).
We found evidence for activation of mitochondrial Ca2+
buffering during and after the inward current through AMPA
receptors, which saturated on repetitive KA application and lead
to a persistent increase in cytosolic Ca2+concentration in motor
neurons, but not in other neurons . Moreover, evidence
exists that excessive entry of Ca2+into motor neurons results in
mitochondrial Ca2+overload leading to depolarization and in
the generation of reactive oxygen species, at least in vitro .
2.1.2. High number of Ca2+-permeable AMPA receptors
Motor neurons appear to have a high proportion of Ca2+-
permeable AMPA receptors (Fig. 3A) and stimulation of these
receptors leads to selective motor neuron death [16,17,56]. In
vitro, we showed that Ca2+entry via Ca2+-permeable AMPA
receptors is responsible for selective motor neuron death .
These cells were killed by a short exposure to KA, while other
neurons were unaffected. This selective motor neuron death was
completely dependent on extracellular Ca2+and was insensitive
to inhibitors of voltage-operated Ca2+or Na+channels. It was
also completely inhibited by the specific AMPA antagonist
LY300164 and by Joro spider toxin, a selective blocker of
AMPA receptors that lack the GluR2 subunit. KA selectively
killed those motor neurons that stained positive for the Co2+
histochemical staining, a measure for the presence of Ca2+-
permeable AMPA receptors. We also quantified the electro-
physiological properties of AMPA receptors dependent on the
relative abundance ofGluR2:sensitivity toexternalpolyamines,
Fig. 3. Difference in Ca2+metabolism between different neurons. The entry pathway and fate of Ca2+is different in motor neurons (A) as compared to other neurons
(B). Motor neurons contain a high amount of Ca2+-permeable AMPA receptors in combination with a low quantity of Ca2+-buffering proteins. As a consequence,
glutamatergic stimulation leads to a high influx of Ca2+. As this Ca2+is not buffered by Ca2+-binding proteins, the cytoplasmic Ca2+concentration will rise and the low
affinity uptake system of mitochondria will start to transport Ca2+into the mitochondria. In non-motor neurons, most AMPA receptors are Ca2+impermeable as they
contain GluR2 and only Na+will enter the cell through these receptors. This will lead to the depolarization of the neuron, to the removal of the Mg2+block at the
NMDA receptor and to Ca2+entry into the cell. Moreover, after depolarization of the cell Ca2+can also enter the neuron through voltage-operated Ca2+channels. In
contrast to motor neurons, in most neurons this Ca2+will be buffered by the Ca2+-binding proteins present in the cytoplasm and Ca2+will not enter the mitochondria.
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rectification index, and relative Ca2+permeability. Motor
neurons had a higher sensitivity to external polyamines, a
lower rectification index, and a higher relative Ca2+permeabil-
ity ratio than other neurons . These findings confirm that
motor neurons are relatively deficient in GluR2. Moreover,
single cell RT-PCR showed that the relative amount of GluR2
mRNA was significantly lower in motor neurons compared to
other neurons, which indicates that the GluR2 regulation
underlying the GluR2 difference is at the transcription level
In vivo evidence for a possible crucial role of GluR2 in
motor neuron viability comes from studies with transgenic
mice. Transgenic mice that lack the GluR2 subunit do not
suffer from a motor neuron disease . This suggests that a
low GluR2 level is not sufficient to cause ALS, but it could
be a modifier of motor neuron degeneration in ALS. In line
with this, we observed that GluR2 deficiency accelerated
significantly the motor neuron degeneration and shortened the
life span of mutant SOD1G93Amice with 15% . Providing
motor neurons with extra GluR2 subunits did also not induce
a phenotype. Crossbreeding these GluR2 overexpressing mice
with mutant SOD1 mice resulted in a 14% increase of the life
span of these double transgenic mice . All together, these
data confirm that the expression level of GluR2 is an
important parameter determining the life span of mutant
The importance of edited GluR2 in neuronal survival is also
indicated by the phenotypes of transgenic mice in which the
extent of RNA editing at the Q/R site is reduced. This generates
a lethal phenotype with seizures and acute neurodegeneration
[73,74]. However, transgenic mice carrying a minigene with the
upstream region of the mouse GluR2 gene that encodes an
asparagine (GluR2-N) at the Q/R site, which makes editing
impossible, are viable and fertile . AMPA receptor channels
incorporating GluR2-N are permeable to Ca2+. The
combined expression of the GluR2-N transgene and endoge-
nous GluR2 alleles caused a moderately (2-fold) increased Ca2+
permeability of the AMPA receptors in neurons of these
transgenic mice . Such transgenic mice were reported to
develop motor neuron degeneration late in their life . More
recently, it was reported that Glu2-N overexpression induced a
progressive decline in the functions of the spinal cord, as well as
late-onset degeneration of spinal motor neurons . Moreover,
crossbreeding heterozygous GluR2-N and mutant SOD1 mice
aggravated the course of the motor neuron disease in the mutant
SOD1 mice and reduced survival by 8% .
2.1.3. AMPA receptor mediated excitotoxicity is aggravated by
In cultured rat spinal motor neurons, chloride influx
aggravated Ca2+-dependent AMPA receptor mediated motor
neuron death . The membrane depolarization caused by
AMPA receptor stimulation resulted in Cl−influx through 5-
nitro-2(3-phenylpropyl-amino) benzoic acid- and niflumic acid-
sensitive Cl−channels. This Cl−influx aggravated excitotoxic
motor neuron death by two mechanisms. It increased the AMPA
receptor conductance and it also resulted in an elevation of the
Ca2+driving force through a partial repolarization. As a
consequence, Cl−ions could play a vital role in glutamate-
mediated excitotoxicity. Although Cl−influx suppresses
neuronal excitability and thereby counteracts the action of
excitatory neurotransmitters such as glutamate, Cl−influx
during exposure to pathological amounts of glutamate could
amplify the excitotoxic action of glutamate on motor neurons.
Co-administration of GABA enhanced the Cl−influx during
AMPA receptor stimulation and this resulted in an increased
Ca2+influx and enhanced cell death, suggesting that concom-
itant GABAergic stimulation may aggravate excitotoxic motor
neuron death . This effect of Cl−influx on excitotoxicity
does not seem to be unique to motor neurons, as similar results
were found in cerebellar granule cells . However, even if it
is a general mechanism it could have more pronounced effects
on motor neurons as they contain a high number of AMPA
receptors  in combination with a very high inhibitory
GABAergic innervation . Moreover, a disturbance of the
GABAergic function was proposed as the mechanism contrib-
uting to excitotoxic upper motor neuron death. Pathological
studies support the loss of cerebral inhibitory interneuronal cells
in ALS  and an alteration in the expression of GABAA
receptor unit subtypes in the motor cortex was reported . In
addition, clinical neurophysiology using transcranial magnetic
stimulation (TMS) has revealed changes in cortical excitability
[83,84]. These hyperexcitability changes reflect both degener-
ation of the corticomotoneuronal system as well as a loss of
inhibition due to an impaired function of inhibitory interneu-
ronal circuits in the motor cortex that renders the motor neurons
hyperexcitable. In conclusion, these data indicate that both a
normal GABAergic stimulation as well as the loss of inhibitory
GABAergic innervation could contribute to excitotoxic motor
2.1.4. Role of metabotropic glutamate receptors
Metabotropic glutamate receptors (mGluRs) appear to
modulate excitotoxicity in motor neurons, but their exact role
remains to be elucidated. The expression pattern of mGluRs
differs between motor neuron populations that are vulnerable
and motor neuron populations that are resistant in ALS. Spinal
and hypoglossal motor neurons mainly express mGluR1, 4 and
7, while motor neurons from Onuf's, oculomotor and trochlear
nucleus also express mGluR5 [85–88]. Stimulation of mGluRs
has been shown to attenuate excitotoxic motor neuron death in
vitro [89,90]. Furthermore, in spinal cords from familial and
sporadic ALS patients an upregulation of mGluRs in reactive
astrocytes has been described . These findings suggest a
regulating role for mGluRs in motor neurons and surrounding
astrocytes, but at present the impact of these findings remains
2.2. Classical excitotoxicity and ALS
2.2.1. Exogenous excitotoxins cause motor neuron disease
Specific forms of motor neuron disease are caused by oral
intake of excitotoxins. This does not only support the
hypothesis that an increase of the glutamatergic stimulation
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can cause neuronal death, but also confirms that motor neurons
are particularly sensitive to this over-stimulation (cfr supra).
Domoic acid concentrated in mussels was responsible for a
food poisoning in Canada due to the consumption of
contaminated mussels from Prince Edward Island [91,92].
This led to gastrointestinal and neurological symptoms. Patients
developed headache, seizures, hemiparesis, ophtalmoplegia and
altered conscious level. In many patients, there was evidence for
a pure motor or sensorimotor neuropathy .
Prolonged consumption of the chickling pea Lathyrus
sativus caused lathyrism . This is a toxic upper motor
neuron disease characterized by spasticity mainly in the lower
limbs. The excitotoxin BOAA (β-N-oxalyl-amino-L-alanine),
which is a potent AMPA receptor agonist  and abundantly
present in this pea, causes this motor neuron degeneration.
Monkeys fed with Lathyrus sativus or BOAA developed upper
motor neuron signs [93,95] reminiscent of lathyrism, and
intrathecal injections with BOAA induced motor neuron
degeneration in rat and mouse spinal cord [42,96].
Western Pacific amyotrophic lateral sclerosis-parkinsonism-
dementia (Guam ALS) is a motor neuron syndrome originally
thought to be caused by the excitotoxin BMAA (β-methyla-
mino-L-alanine) present in the seeds of false sago palm Cycas
circinalis . BMAA is not only an NMDA receptor agonist
, but also activates AMPA receptors . Oral administra-
tion of BMAA to monkeys resulted in a motor neuron disorder,
parkinsonian features and behavioral anomalies with predom-
inantly degenerative changes in the motor cortex and ventral
spinal cord . This finding suggests that excitotoxicity is
involved in the pathogenesis of Guam ALS. However,
alternative mechanisms have been suggested as the concentra-
tion of BMAA in cycad flour in the Western Pacific is low and
Guam ALS can develop many years after the last exposure to
this flour . A recent study suggests that human
consumption of flying foxes, which consumed palm seeds and
possibly accumulated the plant toxins, could be the source of
2.2.2. Detection of increased concentrations of endogenous
excitotoxins in ALS
Crucial to classical excitotoxicity is that the extracellular
concentration of glutamate is increased (Fig. 4). As a
consequence, detection of an increased glutamate concentration
in the extracellular compartment of ALS patients would be a
strong argument. Abnormalities in the glutamate metabolism
have indeed been observed in these patients. Reductions of
glutamate (and aspartate) levels were found in brain and spinal
cord tissue of ALS patients [103–107]. Similarly, levels of
NAAG (N-acetylaspartylglutamate), an acidic dipeptide which
can be converted to glutamate and NAA (N-acetylaspartate) by
NAALADase (N-acetylated-α-linked-amino dipeptidase), and
NAA was reduced, while NAALADase was elevated in brain
and spinal cord tissue of ALS patients [104,105]. In the medulla
of ALS patients evidence was found using proton magnetic
resonance spectroscopy that levels of NAA and NAAG were
reduced, while glutamate levels were increased . In the
cortex of mutant SOD1G93Amice, elevated levels of extracel-
lular glutamate were found in a microdialysis study .
Elevated levels of glutamate (and NAAG, NAA and aspartate)
in the cerebrospinal fluid (CSF) of ALS patients have been
detected in several [105,110], but not in all studies .
Elevated plasma levels of glutamate duringfasting and after oral
Fig. 4. Mechanisms of classical excitotoxicity in ALS. (A) Under normal conditions, the glutamate released by the presynaptic neuron stimulates the Ca2+-permeable
AMPA receptors present in the postsynaptic motor neuron. This leads to a moderate, non-toxic increase of the cytoplasmic Ca2+concentration. The action of glutamate
is terminated by reuptake into the astrocytes surrounding the synapse. (B) If the glutamate transporter is damaged, the reuptake of glutamate into the astrocytes is
diminished, and the concentration of glutamate into the synaptic cleft starts to increase, leading to a higher and/or longer stimulation of the Ca2+-permeable AMPA
receptors. The damage to the EAAT2/GLT1 transporter can be due to the presence of mutant (mt) SOD1 but as the decrease in uptake capacity is also observed in
sporadicALS cases, other unknown factors must be involved.Moreover, activated microglial cells can secretesubstances that increase the glutamatergicstimulation of
the postsynaptic neuron. All together, this results in an increased influx of Ca2+in the motor neurons. As these neurons are devoid of Ca2+-binding proteins,
mitochondria start to buffer Ca2+. (C) A further increase in both the factors released by the microglial cells and in the extracellular glutamate concentration, leads to a
further increase in the cytoplasmic Ca2+concentration in the postsynaptic neuron. This leads to the generation of toxic reactive oxygen species in the mitochondria.
Once the buffering capacity of the mitochondria is exceeded, intracellular Ca2+over-activates different types of Ca2+-dependent enzymes. This interferes with the
normal neuronal function and ultimately leads to neuronal death. The intracellular glutamate content is released from these injured neurons and this results in a
dramatic increase of the extracellular glutamate concentration and in the spread of neurodegeneration.
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glutamate loading were reported in one study , but could
not be confirmed by others [110,111]. A similar controversy
exists about the glutamate dehydrogenase (GDH) activities in
ALS. GDH, a key enzyme in the glutamate metabolism,
reversibly converts 2-oxoglutarate into glutamate. GDH
activities in leukocytes from ALS patients were found to be
decreased in one study , but normal in another . GDH
activities measured in the lumbar spinal cord from ALS patients
were increased in several spinal cord areas, but normal in the
ventral horns . Interestingly, a recent report described a
transgenic mouse model in which the expression of a GDH gene
(GLUD1) was increased. These mice show tremors and hind
limb weakness and a loss of large motor neurons in the ventral
spinal cord .
More consistent evidence indicates that plasma and CSF of
ALS patients contain toxic components that can induce
excitotoxicity [115,116]. Couratier et al.  showed that
CSF from ALS patients was toxic for cortical neurons in
culture and that this neuronal death could be inhibited by the
AMPA receptor blocker CNQX, while NMDA receptor
antagonists had no effect. A recent study reported toxicity
of ALS-CSF to cultured motor neurons . This treatment
substantially elevated the intracellular Ca2+concentration in
motor neurons via AMPA type of glutamate receptors .
It was also shown that CSF from ALS patients can activate
microglia and that toxins released by these activated cells
stimulate ionotropic glutamate receptors leading to the death
of spinal motor neurons  (Fig. 4). Minocycline inhibits
the microglial activation and provides protection against this
CSF toxicity [119,120]. Moreover, we and others have shown
that this semi-synthetic tetracycline analogue influences
disease progression and survival of mutant SOD1 mice
[121–123], indicating that a similar process could be involved
2.2.3. Decreased clearance of glutamate
Rothstein et al.  reported diminished glutamate
transport in synaptosomes from affected brain areas and
spinal cord of sporadic ALS patients. In addition, the number
of [3H]D-aspartate binding sites, a measure for the density of
glutamate transporters, was reduced in spinal cords from
sporadic ALS patients . This decreased glutamate
transport was found to be due to a selective loss of the
astroglial glutamate transporter, EAAT2/GLT1, in the motor
cortex and spinal cord . This loss of EAAT2/GLT1 was
not only observed in sporadic ALS patients [126–128], but
also in familial cases .
AMPA receptor-mediated excitotoxicity of motor neurons
induced by a loss of EAAT2/GLT1 in ALS-affected areas of the
CNS is an attractive hypothesis (Fig. 4). Both in vitro and in
vivo experiments demonstrated that loss of EAAT2/GLT1
function may give rise to selective motor neuron degeneration.
Treatment of organotypic spinal cord cultures with antisense
oligonucleotides to EAAT2/GLT1, resulting in diminished
transporter protein expression, was shown to induce progressive
motor neuron loss, which could be prevented by an AMPA
receptor antagonist . Chronic intraventricular administra-
tion of these antisense oligonucleotides in rats resulted in a rise
of extracellular glutamate levels and a progressive motor
syndrome . A loss of EAAT2/GLT1 immunoreactivity has
also been described in the ventral horn of mutant SOD1G93A
and mutant SOD1G37Rmice [130,131] and more recently in
mutant SOD1G93Arats . However, this finding could not be
reproduced by others [132,133], who found no change in
EAAT2/GLT1 expression or only a retarded mobility on gels.
The mechanism behind the loss of EAAT2/GLT1 protein has
been the subject of intense research, at the DNA, mRNA and
protein level. Mutations in the EAAT2/GLT1 gene have been
looked for, but no clear disease-associated mutations were
found. Interestingly, in one sporadic ALS patient a point
mutation in exon 5 of EAAT2/GLT1 was detected . This
N206S mutation resulted in deficient glycosylation of the
protein and lower EAAT2/GLT1 expression in the cell
membrane . As a consequence, it gave rise to a loss of
glutamate transport capacity. In addition, mutated EAAT2/
GLT1 exerted a dominant negative effect on wild-type EAAT2/
GLT1. In two patients with familial ALS not linked to SOD1, a
mutation in intron 7 and a silent G to A transition in exon 5 of
EAAT2/GLT1 were found . No such mutations were
detected by others [136,137] and therefore their significance
awaits further clarification.
At the mRNA level, an explanation for the loss of EAAT2/
GLT1 could not be identified either. No reductions in EAAT2/
GLT1 mRNA levels were found . Aberrant transcripts of
EAAT2/GLT1 in disease affected areas were reported in
sporadic ALS patients .
Interestingly, a link between ALS-causing SOD1 mutations
and a decreased EAAT2/GLT1 function was found in vitro
: catalysis of H2O2by mutant SOD1 induced oxidative
damage to the intracellular carboxyl-terminal part of EAAT2/
GLT1, resulting in decreased glutamate transport (Fig. 4).
Crossbreeding EAAT2/GLT1 overexpressing mice with mutant
SOD1 mice delayed disease onset as measured by grip strength,
but did not prolong survival .
Recently, β-lactam antibiotics were discovered as potent
stimulators of EAAT2/GLT1 expression after a blinded
screen of 1040 FDA approved drugs and nutritionals
. Both, in vitro and in vivo administration of
ceftriaxone, one of these β-lactam antibiotics, led to a 3-
fold increase in protein levels and a comparable increase in
EAAT2/GLT-1 specific biochemical and electrophysiological
glutamate transport. The mechanism of this overexpression is
the activation of the EAAT2/GLT1 promoter. Ceftriaxone
treatment of mutant SOD1 mice starting at the onset of the
disease significantly increased the life span of the mutant
SOD1 mice from 122 to 132 days. When the drug was
given somewhat earlier, a maximal prolongation of the life
span of the mutant SOD1 mice by 11% was observed .
Moreover, it was shown that ceftriaxone prevented motor
neuron loss and astrogliosis . In conclusion, these data
indicate that an induction of the EAAT2/GLT1 expression
and a higher clearance of glutamate from the synaptic cleft
can protect motor neurons during ALS, at least in the mutant
SOD1 mouse model.
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ARTICLE IN PRESS
2.3. Slow excitotoxicity and ALS
2.3.1. Presence of mutant SOD1 increases sensitivity of motor
neurons to excitotoxicity
In vitro, the sensitivity of motor neurons to the toxicity of
mutant SOD1 was studied by intranuclear injections of mutant
SOD1 in mixed motor neuron cultures . These injections
inducedformation of SOD1 aggregates and motor neuron death,
while no aggregates or cell death was observed after injection in
other cultured neurons. This selective motor neuron death was
dependent on extracellular glutamate as it could be inhibited by
both a general AMPA receptor antagonist (6-cyano-7-nitroqui-
noxaline-dione disodium; CNQX) and a selective antagonist of
Ca2+-permeable AMPA receptors (joro spider toxin) . A
crucial role for intracellular Ca2+in this process was further
indicated by the protection offered to mutant SOD1-induced
motor neuron death by coexpression of the Ca2+-binding
protein, calbindin D28K . These results indicate that the
selective toxicity of mutant SOD1 to motor neurons is induced
or at least can be aggravated by glutamatergic stimulation. The
exact mechanism is not yet clear, although it was suggested that
Ca2+influx through Ca2+-permeable AMPA receptors could be
responsible for aggregation of mutant SOD1 and subsequent
motor neuron death . In a different in vitro model, motor
neurons in mixed spinal cord cultures from SOD1G93Amice
were shown to be more sensitive to glutamate, which resulted in
a higher free radical production . In contrast, we and others
could not find an enhanced sensitivity of cultured motor
neurons overexpressing mutant SOD1 to excitotoxicity
In vivo, treatment protocols that interfered with excitotoxi-
city delayed selective motor neuron death and prolonged
survival in the SOD1G93Amice. The AMPA receptor antago-
nists RPR119990 , 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-
benzo[f]quinoxaline-7-sulfonamide (NBQX, ) and
ZK187638  increased the life span of these mice by
13%, 10% and 11%, respectively. Moreover, interference with
glutamate metabolism by inhibiting glutamate carboxypepti-
dase II with 2-MPPA also increased significantly (by 15%) the
life span of the late onset SOD1G93Amouse model .
Furthermore, it was shown that the motor neuron degeneration
induced by intrathecal administration of the excitotoxin β-N-
oxalylamino-L-alanine (BOAA) was more pronounced in spinal
cords of mutant SOD1G93Amice than in those of non-transgenic
mice . All together, these results strongly indicate that
excitotoxicity plays a role in mutant SOD1 induced motor
neuron death and suggest that SOD1 mutations may render
motor neurons more sensitive to excitotoxicity.
2.3.2. Mutant SOD1 and mitochondrial function
As mentioned above, slow excitotoxicity has often been
linked to mitochondrial dysfunction. As a consequence, it is
interesting to note that there is growing evidence that mutant
SOD1 is not only localized in mitochondria, but also interferes
with their function (Fig. 5). The presence of human mutant
SOD1 in vacuolated mitochondria was first demonstrated by
electron microscopy in mutant SOD1G93Atransgenic mice
activity of several mitochondrial respiratory chain complexes
was reduced and that ATP synthesis was diminished in mutant
SOD1G93Amice. These deficits in mitochondrial function
became more pronounced with progression of the disease and
affected the spinal cord more than other tissues. This
mitochondrial dysfunction caused by the presence of mutant
SOD1 mayhave many consequences. First, itispossiblethat the
localization of mutant SOD1 in the mitochondria on itself is
pathogenic(independent ofexcitotoxicity). WhenmutantSOD1
was targeted to different subcellular compartments, targeting it
Fig. 5. Mechanisms of slow excitotoxicity in ALS. (A) Factors and growth factors released from the astroglial cells surrounding the synapse activate the expression of
the GluR2 subunit in the motor neuron. As a consequence, the number of Ca2+-permeable AMPA receptors in the postsynaptic neuron is controlled and stimulation of
the AMPA receptors only leads to a moderate influx of Ca2+through this type of receptors. (B) If the release of the astroglial factor is diminished, the number of Ca2+-
permeable AMPA receptors is increased and the influx of Ca2+into the postsynaptic neuron increases. Moreover, reduced editing of the mRNAs encoding the GluR2
subunit can also be responsible for an increase in the Ca2+permeability of the AMPA receptors. As the presence of mutant (mt) SOD1 compromises the Ca2+-buffering
capacity of the mitochondria, the intracellular Ca2+concentration in the cytoplasm of the postsynaptic neuron raises. (C) A dramatic increase in the Ca2+permeability
of the AMPA receptors in combination with a drastic decrease in the Ca2+-buffering capacity of the mitochondria leads to an excessive activation of different types of
Ca2+-dependent enzymes. As in classical excitotoxicity, this interferes with the normal neuronal function and ultimately leads to neuronal death.
8 L. Van Den Bosch et al. / Biochimica et Biophysica Acta xx (2006) xxx–xxx
ARTICLE IN PRESS
to the mitochondria was sufficient to induce cell death in a
neuronal cell line . It was shown that the presence of
mutant SOD1 in the mitochondria triggered the release of
cytochrome c followed by the activation of the caspase cascade
Second, mitochondrial dysfunction makes it difficult for the
affected neuron to meet the demands of its many ATP-
dependent processes. The delicate ion balance thus becomes
disturbed and may give rise to slow excitotoxicity and lead to
cell death of neurons with a high glutamatergic input, such as
motor neurons. Moreover, the damage caused by mutant SOD1
on mitochondria may also be more pronounced in these motor
neurons as these cells have a high metabolic demand.
Last but not least, dysfunction of mitochondria will also
have a dramatic effect on the mitochondrial Ca2+-buffering
capacity of these organelles . This defect will have more
pronounced consequences in the motor neurons as these
organelles play a crucial role in the Ca2+buffering of these
cells (cfr supra). As a consequence, the cytoplasmic Ca2+
concentration rises and over-activates a number of Ca2+-
dependent enzymes and this could lead to neuronal death
2.3.3. Reduced editing of the GluR2 mRNA
Under normal conditions, all the GluR2 mRNA is edited
resulting in the change of a CAG codon into a CGG codon. If
this edited GluR2 subunit is incorporated in the AMPA receptor,
it is Ca2+impermeable. However, it was demonstrated that the
editing efficiency of GluR2 mRNA was lower in the ventral
spinal gray matter of ALS patients compared to controls .
This deficiency in GluR2 editing was confirmed in single motor
neurons isolated with laser microdissection from five ALS
patients . The consequence of this editing deficiency is that
AMPA receptors that contain GluR2 translated from unedited
mRNA will have a neutral glutamine instead of the positively
charged arginine and will be Ca2+permeable (Fig. 5).
Apparently, this phenomenon is limited to sporadic ALS
patients as it was not found in mutant SOD1G93Amice (our
unpublished results) or in mutant SOD1G93Arats .
2.3.4. Astrocytes affect sensitivity of motor neurons to
There is increasing evidence that the selective motor neuron
death in mutant SOD1-dependent motor neuron death is a non-
cell-autonomous process . Cells surrounding the motor
neuron that do not contain mutant SOD1 can protect mutant
SOD1 containing motor neurons and vice versa. Moreover, it is
generally accepted that astrocytes are crucial to keep the
synaptic glutamate concentration very low (cfr supra). We
recently discovered that astrocytes can also influence the
subunit composition of the AMPA receptor as they determine
the GluR2 expression in motor neurons (Fig. 5). We found that
two rat strains differed in their sensitivity to transient spinal cord
ischemia induced by clamping the aortic arch and left
subclavian artery. This process was mediated by AMPA
receptor stimulation, as it was completely prevented by pre-
treatment with the AMPA receptor antagonist NBQX. The
motor neuron degeneration induced by ischemia was also
clearly different in the two different rat strains. This difference
in the sensitivity to excitotoxicity was preserved in cultured
spinal motor neurons on top of a astrocytic feeder layer isolated
from these two different rat strains [160,161]. Motor neurons
cultured from the rat strain with the lowest sensitivity to
transient ischemia expressed AMPA receptors with a low Ca2+
permeability and a high GluR2 expression . Remarkably,
the astrocytic feeder layer on which these motor neurons were
cultured determined these characteristics. When motor neurons
were shifted from one astrocytic feeder layer to the other, they
changed their characteristics according to the origin of the
astrocytes. . We conclude from these experiments that
factors released from astrocytes can influence the sensitivity of
motor neurons to excitotoxicity by influencing the expression
level of GluR2 and the relative amount of Ca2+-permeable
AMPA receptors. Moreover, it seems that the presence of
mutant SOD1 in the astrocytes can affect the secretion of these
factors, leading to a lower expression of GluR2, more Ca2+
permeable AMPA receptors and a higher sensitivity to
excitotoxicity (unpublished results).
Further evidence for an effect of glial cells on the
characteristics of the AMPA receptors comes from the
observation that both glial derived neurotrophic factor
(GDNF) as well as brain derived neurotrophic factor (BDNF)
enhance GluR2 expression. The GluR2 promoter contains a
neuron-restrictive silencer element (NRSE). Binding of neuron-
restrictive silencing factor (NRSF) to this sequence in non-
neuronal cells results in silencing of genes with a NRSE in their
promoter [162–164]. It was shown that the effect of GDNF on
the GluR2 promotor was mediated by NRSE . Delivery of
GDNF to motor neurons with adeno-associated viral or
adenoviral vectors resulted in an increase of the life span of
mutant SOD1G93Amice by 14% [166,167]. Whether GDNF-
induced upregulation of GluR2 and/or interference with
excitotoxicity contributed to this protective effect was not
2.3.5. Interference with axonal transport
As motor neurons have extremely long axons, interference
with axonal transport will have dramatic consequences on the
functioning of these cells. Moreover, a pathological hallmark of
ALS is the abnormal accumulation of neurofilaments in the
perikaryon and in the axons of motor neurons [168,169].
Although it is not yet clear how these accumulations contribute
to the neurodegenerative process, their presence suggests that
axonal transport is disturbed in affected motor neurons. An
interesting link between excitotoxicity and axonal transport was
found in primary cortical neurons. After treatment of these
neurons with glutamate, a dose-dependent slow down of the
anterograde axonal transport was observed . This
phenomenon was accompanied by phosphorylation of the
neurofilament side-arm domains by different members of the
mitogen-activated protein kinase family . Although, this
phenomenon was dependent on the stimulation of NMDA
receptors, a similar process involving (excessive) stimulation of
the Ca2+-permeable AMPA receptors could have comparable
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ARTICLE IN PRESS
effects in motor neurons, although experimental evidence for
this attractive hypothesis is lacking.
Since its description by Charcot more than 130 years ago, the
pathogenesis of selective motor neuron degeneration in
amyotrophic lateral sclerosis (ALS) remains unresolved. Over
the years, many pathogenic mechanisms have been proposed.
Amongst others these include: oxidative stress, aggregate
formation of mutant SOD1, inflammation, growth factor
deficiency, neurofilament disorganisation and last but not least
excitotoxicity. This multitude of contributing factors indicates
that ALS is a complex disease and also suggests that ALS is a
It is obvious that not everything in the pathogenesis of ALS
can be reduced to the involvement of excitotoxicity, but it is
more and more clear that at least some of the pathogenic
mechanisms are interconnected. For instance, more and more
evidence indicates that excessive stimulation of glutamate
receptors could lead to Ca2+overload in mitochondria resulting
in the overproduction of reactive oxygen species and to
oxidative stress. Moreover, motor neurons could become
more sensitive to glutamatergic (over)-stimulation and to
excitotoxicity due to the presence of mutant SOD1 in the
mitochondria. Another phenomenon that could sensitize motor
neurons is inflammation and concomitant microglial activation.
Last but not least, the subunit composition of the AMPA
receptor seems to be dependent on growth factors and/or on
other factors released by the surrounding astrocytes. Shortage of
these factors could result in motor neurons with a higher number
of Ca2+-permeable AMPA receptors that are more sensitive to
Apart from being (part of) the pathogenic mechanism
leading to ALS, excitotoxicity could also be responsible for
the selective vulnerability of motor neurons during the course
of the disease. In contrast to most other neurons, motor
neurons have a low Ca2+-buffering capacity due to the low
expression of Ca2+-buffering proteins and a high number of
Ca2+-permeable AMPA receptors resulting from a low
expression of the GluR2 subunit. The combination of these
two properties seems to be intrinsic to motor neurons and is
most likely essential for their normal function. However,
under pathological conditions, motor neurons could become
over-stimulated by glutamate and overwhelmed with Ca2+.
An immediate consequence of the lack of Ca2+-buffering
proteins is that mitochondria become important for Ca2+
buffering. Whether the downstream pathways activated by the
intracellular Ca2+increase are different in motor neurons
compared to other neurons is not yet known.
It is not yet clear whether all this knowledge about
excitotoxicity and motor neurons could result in a therapy
with a benefit for the ALS patient of more than a few months,
obtained with riluzole to date. Combining this drug, that
interferes with glutamate release, with a selective inhibitor of
the Ca2+-permeable AMPA receptors could be a valuable
option. Another option is to increase the resistance of motor
neurons to high intracellular Ca2+concentrations by inducing
defence mechanisms and/or to inhibit the downstream pathways
activated by an increased intracellular Ca2+concentration.
Moreover, recent research indicates that therapeutic options do
not have to focus on motor neurons alone, as ALS seems to be a
non-cell-autonomous disease. As a consequence, inhibiting
microglial activation which prevents the release of toxic
substances by these cells could also be a valuable option. In
addition, stimulating astrocytes to increase glutamate uptake
and/or to secrete (growth) factors that modify the properties of
the AMPA receptors present on the motor neurons could also
have a positive effect. In conclusion, a combined pharmaco-
logical interference with the many faces of excitotoxicity both at
the motor neurons and at the surrounding cells will be most
likely essential to extend survival of ALS patients.
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