Glutamate Transporters and the Excitotoxic Path to Motor Neuron Degeneration in Amyotrophic Lateral Sclerosis

Abstract

Responsible for the majority of excitatory activity in the central nervous system (CNS), glutamate interacts with a range of specific receptor and transporter systems to establish a functional synapse. Excessive stimulation of glutamate receptors causes excitotoxicity, a phenomenon implicated in both acute and chronic neurodegenerative diseases [e.g., ischemia, Huntington's disease, and amyotrophic lateral sclerosis (ALS)]. In physiology, excitotoxicity is prevented by rapid binding and clearance of synaptic released glutamate by high-affinity, Na(+)-dependent glutamate transporters and amplified by defects to the glutamate transporter and receptor systems. ALS pathogenetic mechanisms are not completely understood and characterized, but excitotoxicity has been regarded as one firm mechanism implicated in the disease because of data obtained from ALS patients and animal and cellular models as well as inferred by the documented efficacy of riluzole, a generic antiglutamatergic drug, has in patients. In this article, we critically review the several lines of evidence supporting a role for glutamate-mediated excitotoxicity in the death of motor neurons occurring in ALS, putting a particular emphasis on the impairment of the glutamate-transport system.

Full text

Forum Review Article
Glutamate Transporters and the Excitotoxic Path to Motor
Neuron Degeneration in Amyotrophic Lateral Sclerosis
Emily Foran and Davide Trotti
Abstract
Responsible for the majority of excitatory activity in the central nervous system (CNS), glutamate interacts with a
range of specific receptor and transporter systems to establish a functional synapse. Excessive stimulation of
glutamate receptors causes excitotoxicity, a phenomenon implicated in both acute and chronic neurodegener-
ative diseases [e.g., ischemia, Huntington’s disease, and amyotrophic lateral sclerosis (ALS)]. In physiology,
excitotoxicity is prevented by rapid binding and clearance of synaptic released glutamate by high-affinity, Na
þ
-
dependent glutamate transporters and amplified by defects to the glutamate transporter and receptor systems.
ALS pathogenetic mechanisms are not completely understood and characterized, but excitotoxicity has been
regarded as one firm mechanism implicated in the disease because of data obtained from ALS patients and
animal and cellular models as well as inferred by the documented efficacy of riluzole, a generic anti-
glutamatergic drug, has in patients. In this article, we critically review the several lines of evidence supporting a
role for glutamate-mediated excitotoxicity in the death of motor neurons occurring in ALS, putting a particular
emphasis on the impairment of the glutamate-transport system. Antioxid. Redox Signal. 11, 1587–1602.
Glutamate in the Central Nervous System
L-Glutamate is the predominant excitatory neurotrans-
mitter in the central nervous system (CNS). A nonessen-
tial amino acid, glutamate is continuously converted to
a-ketoglutarate through deamination by glutamate dehydro-
genase or by transamination by one of the transaminases and
metabolized through the tricarboxylic acid cycle to succinate,
fumarate, and malate, successively. Glutamate is also the
product of the deamination of glutamine by phosphate-
activated glutaminase, a mitochondrial and possibly neuron-
specific enzyme (80). Synaptically released glutamate activates
a family of ligand-gated ion channels (ionotropic receptors)
and G protein–coupled receptors (metabotropic receptors),
and its action is terminated by specific reuptake systems lo-
cated mainly in astrocytes surrounding the synapse. In astro-
cytes, glutamate is then converted into glutamine, which does
not have neurotransmitter properties and can be released and
made available for neurons to convert it back to glutamate
through a glutamine-reuptake system. Glutamate is then
packed by vesicular glutamate transporters in synaptic vesi-
cles, ready to be released again (35, 129) (Fig. 1).
Three major classes of metabotropic and three of ionotropic
receptors for glutamate are known (Table 1). Both receptor
families localize to different structures of the excitatory syn-
apse, including the presynaptic terminal and the postsynaptic
element, and astrocytes that envelop the synapse (75, 100).
The ionotropic receptor complexes are classified according
to their responsiveness and affinity to exogenous agonists;
N-methyl-d-aspartic acid (NMDA), a-amino-3-hydroxy-5-
methyl-4-isoxazolepropionic acid (AMPA), and kainate
(KA). Classically, a demarcation existed between the Ca
2þ
-
permeable NMDA receptors and the Ca
2þ
-impermeable
AMPA and KA receptors. However, AMPA receptors missing
the GluR2 subunit have been shown to be Ca
2þ
permeable (70,
152). GluR2-deficient AMPA receptors are expressed in the
motor neurons and are implicated in excitotoxic degeneration
(34, 73, 145).
The levels of glutamate in the mammalian CNS are very
high compared with the levels of all other neurotransmitters,
ranging between 5 and 10 mmol=kg of tissue (22). Excito-
toxicity is caused by the excessive and dysregulated activation
of glutamate receptors. Prolonged exposure of these receptors
to high or persistently increased concentrations of glutamate
Weinberg Unit for ALS Research, Farber Institute for the Neurosciences, Thomas Jefferson University, Philadelphia, Pennsylvania.
ANTIOXIDANTS & REDOX SIGNALING
Volume 11, Number 7, 2009
ªMary Ann Liebert, Inc.
DOI: 10.1089=ars.2009.2444
1587

can lead the cell expressing these receptors to death (27). In
physiologic conditions, extracellular levels of glutamate are
maintained at submicromolar concentrations, more likely in
the nanomolar concentration range (64), which is too low to
cause activation of the high-affinity glutamate receptors.
During synaptic release events, glutamate concentration can
increase up to the millimolar range (32). Excitotoxicity is
propagated primarily through the Ca
2þ
-permeable receptors.
Influx of Ca
2þ
is buffered by the endoplasmic reticulum (ER)
and the mitochondria, but in the presence of excess Ca
2þ
in-
flux, these systems can be overwhelmed. Ca
2þ
overload or
perturbations of intracellular Ca
2þ
compartmentalization can
activate or enhance mechanisms leading to cell death. An
imbalance between Ca
2þ
influx and efflux from cells is the
initial signal leading to Ca
2þ
overload and death of neurons
(Fig. 1). In addition, alterations in intracellular Ca
2þ
storage
can integrate with death signals that do not initially require
Ca
2þ
, to promote processing of cellular components and
death by apoptosis or necrosis. Finally, Ca
2þ
can directly ac-
tivate catabolic enzymes such as proteases, phospholipases,
and nucleases that directly cause cell demise and tissue
damage. When the mitochondrial buffering system fails, the
cell becomes highly vulnerable to mitochondria-mediated
apoptosis, reactive oxygen species (ROS), production and
electron-chain dysfunction (30, 43).
Glutamate is cleared from the intersynaptic milieu by
specialized transporters for a normal, nonpathogenic func-
tioning of the synapses (69). Unlike acetylcholine at the neu-
romuscular junction, which is enzymatically degraded, no
evidence exists for the presence of extracellular synaptic en-
zymes that can inactivate glutamate (69). Clearance of gluta-
mate is accomplished by a family of glutamate-transporter
proteins. Five high-affinity, Na
þ
-dependent glutamate trans-
porters have been identified and termed EAAT1–5, also
known in rodents as GLAST, GLT-1, EAAC1, and rodent
EAAT4–5 (Table 2). These transporters share *50–60% amino
acid sequence similarity and have varying cellular and ana-
tomic distributions (6, 18, 72). EAAT3–5 are expressed by
neurons throughout the brain. Notably, EAAT4 and EAAT5
are specifically located in Purkinje cells in the cerebellum and
the retina, respectively. EAAT1 and EAAT2 are located
mainly on astrocytes, although they also are expressed by
other glia cells like oligodendroglia and macrophages, with
EAAT1 primarily expressed in the cerebellum, and EAAT2
widespread throughout the CNS (36). Splice variants of
EAAT2 have also been cloned, but their abundance is rela-
tively low, and their specific purpose still unclear (25, 26). The
EAATs have structural differences and varying affinity for
glutamate and sensitivities to glutamate-receptor agonists,
which create physiological differences in activity. The KA-
receptor agonist kainic acid, and its dehydrogenated form,
dihydrokainic acid (DHK), specifically block, with high af-
finity, EAAT2, which highlights a significant difference be-
tween the classes of transporters (6). Crystallographic studies
recently shed light on the architecture of glutamate trans-
porters. Yernool and colleagues (156) crystallized a glutamate
transporter (Glt-ph) from the obligate anerobe, Pyrococcus
horikoshii, which shares *40% homology with the eukaryotic
glutamate transporter EAAT2. The protomer structure con-
tains eight transmembrane regions, which are predominantly
a-helical (regions 4 and 7 are segmental a-helices), a large
extracellular loop connecting transmembrane region 3 and 4,
FIG. 1. (A)In a normally functioning synapse, glutamate
released from the presynaptic terminals activates the
NMDA and AMPA receptors, resulting in an influx of Na
and Ca ions into the postsynaptic element, depolarization of
the neuron, and ultimately, an action potential. The neuro-
transmitter action is then terminated by glutamate transport-
ers located in the nearby astroglia cells, as well as in the
postsynaptic elements. (B) Excitotoxicity can be induced by an
elevation of synaptic glutamate concentration. This can be
caused by an increased released of glutamate and=or an im-
paired glutamate uptake. The excessive stimulation of the
glutamate receptors that results from this increased synaptic
glutamate gives rise to an increased intracellular concentration
of Ca ions, resulting in neuronal death. Neuronal cell loss re-
sulting from this process can cause a further increase in ex-
tracellular glutamate and amplifies the excitotoxic damage.
1588 FORAN AND TROTTI

and two hairpin regions. These data, combined with bio-
chemical evidence, also predict that the functional glutamate
transporter has a homotrimeric quaternary structure, which is
conserved in bacterial and human transporters (53, 156).
Glutamate transporters account for the bulk transport of
glutamate across the plasma membrane of cells and act
quickly to buffer synaptically released glutamate (140). To
accomplish these tasks, glia cells express transporters in
abundance, whereas neurons express fewer transporters, al-
though they appear to be precisely located near or at the
synapses. Several lines of evidence suggest that neuronal
transporters work by controlling activation of metabotropic
glutamate receptors at the postsynapse (17) and by limiting
glutamate spillover between adjacent synapses (38) whereas
their glial counterparts function as the main glutamate sinks
for all released glutamate. The glial glutamate transporter
EAAT2 is very abundant in the brain, representing up to 1% of
total brain proteins (35), and it is therefore thought to be
primarily responsible for the removal of glutamate from the
synapse (113). A clear understanding of the contribution of
EAAT2 to total glutamate transport in the CNS came from
studies performed in synaptosomes prepared from EAAT2-
knockout mice. Tanaka and colleagues (130) found that glu-
tamate uptake in cortical crude synaptosomes of EAAT2
(=)
mice was reduced to 5.8% of that measured in synaptosomes
from wild-type mice, indicating that EAAT2 is responsible for
the greatest proportion of glutamate transport in the CNS.
Phenotypically, EAAT2-knockout mice are hyperexcitable
and die prematurely (50.0% survival after 6 weeks) with oc-
currence of spontaneous epileptic seizures and behavioral
patterns similar to those of N-methyl-d-aspartate (NMDA)–
induced seizures, underscoring the role for EAAT2 in main-
taining functional excitatory neurotransmission. By using
long-term antisense oligonucleotide administration, in vitro
and in vivo, Rothstein and colleagues (115) demonstrated that
loss of the glial glutamate transporters EAAT1 or EAAT2
produced elevated extracellular glutamate levels, neurode-
generation characterized by excitotoxicity, and a progressive
paralysis in rats. These studies suggest that glial gluta-
mate transporters could provide the majority of functional
Table 1. Classification of Glutamate Receptors
Receptor class Subunits
Permeability=second
messenger system Antagonist
Special
properties
NMDA (ionotropic) N1 Na
þ
APV Mg
2þ
block
N2a Ca
2þ
Gly coactivator
N2b
N2c
N2d
N3a
N3b
AMPA (ionotropic) GluR1
GluR2
Na
þ
(Ca
2þ
)*
CNQX Q=R editing in
GluR subunit
GluR3
GluR4
Kainate (ionotropic) GluR5 Na
þ
CNQX
GluR6
GluR7
KA1
KA2
Class I (metabotropic) mGluR1, IP
3
,Ca
2þ
LY393675,
mGluR5 MPEP
Class II (metabotropic) mGluR2, cAMP LY341495
mGluR3
Class III (metabotropic) mGluR4, cAMP CPPG
mGluR6,
mGluR7,
mGluR8
*GluR2 dependent.
Both ionotropic and metabotropic receptors comprises three functional defined classes made up of several individual subunits, each
encoded by a different gene. Abbreviations: AMPA, a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; APV, (2R)-amino-5-
5phosphonovaleric acid; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione.
Table 2. Classification and distribution of glutamate
transporters in the nervous system
Human
gene
name
Rodent
gene
name
Cellular
expression
Anatomical
distribution
EAAT1 GLAST Astrocytes Cerebellum
EAAT2 GLT1 Astrocytes Widespread
throughout CNS
EAAT3 EAAC1 Neurons Widespread
throughout CNS
EAAT4 Rodent Neurons Purkinje cells
of the cerebellum
EAAT4
EAAT5 Rodent Neurons Retina
EAAT5
EAAT, excitatory amino acid transporter; GLT, glutamate trans-
porter; GLAST, glutamate-aspartate transporter.
GLUTAMATE TRANSPORTERS IN ALS 1589

glutamate transport and are essential for maintaining low
extracellular glutamate and for preventing chronic glutamate
neurotoxicity.
Changes in expression and activity of glutamate trans-
porters have been reported in many neurodegenerative dis-
eases such as Huntington’s disease, Parkinson’s disease,
Alzheimer’s disease, and amyotrophic lateral sclerosis (ALS),
but also in astrogliomas, epilepsy, and in more-acute neuro-
pathologic events like stroke and ischemia (90). In ALS, the
role of the glutamate transporter EAAT2 has been investi-
gated more thoroughly. In chronic neurodegenerative dis-
eases, however, it is not clear whether these dysfunctions in
the glutamate-transport system contribute to the pathogene-
sis or whether they are more a secondary event consequential
to primary pathologic insults. This is not to say that, in the
latter case, glutamate-transporter dysfunction does not play a
role in the overall pathologic manifestation of the diseases.
However, the temporal correlation between glutamate-
transporter dysfunction and pathology is a question that must
be addressed more thoroughly, as it may have important
therapeutic and mechanistic implications.
Amyotrophic lateral sclerosis and glutamate
ALS is a fatal paralytic disorder characterized by selective
death of motor neurons. Approximately 10% of ALS cases are
inherited (FALS), and 90% are sporadic (SALS). About 25% of
the FALS cases are caused by missense mutations in the
ubiquitously expressed enzyme Cu
2þ
=Zn
2þ
superoxide dis-
mutase (SOD1). The symptoms and pathology of SOD1-FALS
closely resemble the rest of ALS cases, raising considerable
enthusiasm for the transgenic animal models expressing
human SOD1 mutations (mutSOD1), in the hope that these
models could provide insights into the pathogenic mecha-
nisms of both FALS and SALS. MutSOD1-mediated toxicity
results from the impairment of multiple cellular functions (12,
104). Ubiquitous expression of high levels of mutSOD1 causes
progressive motor neuron disease in transgenic mice (i.e.,
SOD1-G93A, G37R, and G85R) and rats (H46R and G93A)
that recapitulates most of the clinical features of human ALS
(20, 60, 68, 98, 154). Although the cause of paralysis in ALS is
the death of motor neurons, the cell autonomy of the patho-
genesis has been questioned by studies in which selective
excision of mutSOD1 from microglia and astrocytes empha-
sized the role of these cells as key contributors in ALS path-
ogenesis (13, 31, 155). Several factors originating from
different cell types were also investigated as potential toxic
molecules that could mediate motor-neuron death (12, 104).
Among these, a role for the dysregulation of glutamate ho-
meostasis in ALS-mediated neurodegeneration has been es-
tablished, based on the following evidence:
1. Motor neurons showed a marked vulnerability to glu-
tamate excitotoxicity (112, 144–147). In vitro experi-
ments showed that motor neurons in spinal cord
organotypic cultures are particularly vulnerable to
increased glutamate levels or to AMPA-receptor–
mediated excitotoxicity (116, 119). Similarly, induction
of motor-neuron death was also achieved by activating
Ca
2þ
-permeable AMPA receptors both in vitro, in a co-
culture system consisting of motor neurons seeded on
an astrocytic monolayer (145), and in vivo by delivery in
the mouse spinal cord of selective agonists. (66, 71, 99,
136).
2. Increased plasma levels of glutamate (1, 107), decreased
glutamate uptake, decreased expression levels of the
glial glutamate-transporter EAAT2 (47, 117), and al-
tered glutamine synthetase (14) have been documented
in ALS patients.
3. Cerebrospinal fluid (CSF) collected from ALS patients,
but not from healthy controls, was shown to cause ex-
citotoxicity in neuronal cultures, which is blocked by
glutamate-receptor antagonists. This implies that the
levels of glutamate released in the extracellular milieu
are higher in patients with ALS (29, 62, 124).
4. The only effective treatment available today for ALS is
the antiglutamatergic drug riluzole, which is routinely
prescribed for ALS patients. Riluzole regulates gluta-
mate release, postsynaptic receptor activation, and in-
hibits voltage-sensitive channels (2, 10). Riluzole also
was found to increase significantly glutamate uptake in
a dose-dependent manner in the mouse CNS, facilitat-
ing the buffering of excessive extracellular glutamate
and suggesting that the neuroprotective action of rilu-
zole might be partly mediated by its activating effect on
glutamate uptake (51). Treatment with riluzole de-
creased the plasma levels of excitatory amino acids
during late stages of ALS in patients (101), although
these data should not be considered conclusive (2). Ri-
luzole remains the only FDA-approved drug for ALS,
based on the 3-month improvement in survival ob-
served in two large clinical trials (10, 82, 83).
A role for astrocytes and impairment of the astroglial
glutamate-transporter EAAT2 in ALS
Astrocytes intimately interact with neurons to provide
trophic support and actively participate in neuronal excit-
ability by controlling the extracellular levels of ions and
neurotransmitters (149). Astrocytes also exert potent trophic
influences on motor neurons through a variety of proteins and
molecules. In response to injury, astrocytes and microglia
display characteristic phenotypic changes characterized as
astrocytosis or gliosis and respond to pathologic stress by
proliferating and adopting a reactive phenotype, which is
characterized morphologically by hypertrophic nuclei and
cell bodies, and elaboration of distinctly long and thick pro-
cesses with increased content of glial fibrillary acidic protein
(GFAP). In addition, reactive astrocytes express a wide vari-
ety of markers, such as cytoskeleton proteins, cell-surface and
matrix molecules, proteases, protease inhibitors, several
growth factors, and cytokines (111). By secreting diffusible
factors, damaged neurons or activated astrocytes interact in a
complex manner with immune cells and microglia. Activated
microglia, in turn, secrete proinflammatory peptides, nitric
oxide (NO), and excitotoxins that further induce astrocytosis
or aggravate neuronal damage, therefore perpetuating and
amplifying a local pathogenic process (56). Recent evidence
indicates the existence of mechanisms by which activated
astrocytes may contribute either to the death of neurons or to
their survival in response to damage (7, 106, 132). Under-
standing these processes and the interaction between neurons
and glia may help to explain the induction and the propaga-
tion of motor-neuron loss in ALS.
1590 FORAN AND TROTTI

Astroglia dysfunction in ALS occurs through different
synergistic mechanisms
Cytokine production by astrocytes. Much of the research
on the pathology of neurodegenerative diseases has been fo-
cused on neuroinflammatory mechanisms. In ALS, neuroin-
flammation involves the entire motor system (63). Important
functional interactions have been described between IL-1b
expression by glial cells and the occurrence of excitotoxic
mechanisms and neuronal death in diverse forms of neuro-
degeneration, which could be relevant in ALS pathophysi-
ology. Interestingly, cytokine signaling can induce iNOS,
COX-2, and NMDA-receptor phosphorylation, with different
consequences in glial and neuronal cells. Activation of iNOS
in astrocytes by IL-1bpotentiates NMDA-mediated neuro-
toxicity in mixed cortical cultures (65).
Production of nitric oxide (NO8) and peroxynitrite
(ONOO

). Free radical damage is a characteristic of ALS
tissues (46). Several reports have shown that reactive astro-
cytes in culture may contribute to free-radicals formation and
neuronal death. In particular, induction of iNOS by lipo-
polysaccharide (LPS) or cytokines seems to be required for
astrocytes to promote neuronal death (126). Barbeito and
colleagues (24, 105) reported that production of NO8by re-
active astrocytes is required for the induction of motor-neuron
apoptosis in a co-culture model. Apoptotic motor neurons
were immunoreactive for nitrotyrosine, suggesting a role for
ONOO

.NO8itself cannot nitrate tyrosine, which implies
that it was transformed into peroxynitrite by reaction with
superoxide. Nitrotyrosine staining has been reported in cul-
tured motor neurons undergoing apoptosis (44, 45), in spinal
cord of mutSOD1 mice, and in sporadic and familial cases of
ALS (46).
Production of apoptotic factors. Cytokines and trophic
factors produced by reactive astrocytes such as FasL, TNF-a,
and NGF, are capable of activating death receptors expressed
in the diseased CNS. Receptor-mediated apoptosis could play
a role in motor neuron loss in ALS without the direct in-
volvement of the immune system. These factors show a dual
function, promoting cell survival or death, depending on gene
expression and activation state of the target cell (i.e., motor
neurons) (8). Another potential apoptotic candidate released
by astrocytes is NGF. Clearly, NGF is critical for the differ-
entiation and survival of specific neuronal populations during
development and for neural plasticity in the mature CNS
(121). Whereas NGF can signal through activation of the high-
affinity TrkA receptor, it also can activate the nonselective
neurotrophin receptor p75
NTR
, a member of the tumor ne-
crosis factor–receptor superfamily. Motor neurons are gen-
erally unresponsive to NGF because they lack the specific
TrkA receptor. Signaling through p75
NTR
, in the absence of
the corresponding Trk receptor, has been shown to promote
apoptosis in specific neuronal types during normal CNS de-
velopment (49) and is probably used to eliminate damaged
neurons and oligodendrocytes in the mature CNS. Motor
neurons express p75
NTR
during the embryonic period of
naturally occurring cell death when more than half of motor
neurons die, but its expression gradually ends after birth.
Although p75
NTR
is not present in mature motor neurons, the
receptor can be re-expressed after nerve injury (110). More-
over, p75
NTR
is found in motor neurons of ALS patients (88),
suggesting that re-expression of the receptor might modulate
the death of neurons in damaged areas. Astrogliosis is asso-
ciated with increased expression and release of several
growth factors and cytokines, including NGF (42). Little is
known about the expression of NGF in ALS, although in-
creased NGF levels were reported in muscle of ALS patients
(127). Thus, it is conceivable that NGF signaling between as-
trocytes and p75
NTR
-expressing motor neurons may contrib-
ute to the induction of neuronal apoptosis in ALS.
Downregulation and impairment of the glutamate trans-
porter EAAT2. The downregulation of EAAT2 expression
and activity levels in ALS suggests a connection between this
disease and synaptic glutamate homeostasis. Expression of
EAAT2 is dramatically decreased in postmortem spinal cord
specimens of ALS patients, particularly in the ventral horn,
where motor neurons are found (86). The first demonstration
of an impaired glutamate-transport system was obtained by
direct measurements of
3
H-l-glutamate uptake in synapto-
somes prepared from different CNS areas of sporadic ALS
patients. The patients displayed a marked decrease in the
maximal transport velocity (V
max
) for glutamate in synapto-
somes prepared from spinal cord (59%), motor cortex
(70%), and somatosensory cortex (39%), but not in syn-
aptosomes prepared from regions not affected by the disease,
such as visual cortex, striatum, or hippocampus, or when
compared with the corresponding regions in unaffected in-
dividuals or other neurodegenerative disease patients (117).
The decrease of glutamate uptake (47, 117) has been linked
specifically to a decrease in the levels of EAAT2 expression
(19, 120). Although clear alterations in EAAT2 levels are
found in patients with ALS, it is not likely that these reduc-
tions could have a genetic cause. With single-strand confor-
mation polymorphism analysis of genomic DNA, Aoki and
colleagues (137) identified one novel mutation in the EAAT2
gene in a single sporadic ALS patient and two novel muta-
tions in two affected familial non-SOD1 ALS siblings. In the
sporadic ALS patient, the mutation substitutes serine for an
asparagine and removes one N-linked glycosylation site in the
EAAT2 protein, affecting the normal function of the trans-
porter. In the two affected individuals in the ALS family, a
mutation in the 5’ end of intron 7 and a silent G ?A transition
at codon 234 in exon 5 was also reported (4). However, no
suggestion has been made that this polymorphism is widely
represented among the ALS population or can cause the dis-
ease.
Abnormal variants of EAAT2 mRNA resulting from in-
correct splicing were found in the affected CNS areas of ALS
patients (86). These intron-retention and exon-skipping
mRNA species encoded truncated EAAT2 fragments thought
to have dominant-negative effects on the expression and ac-
tivity of EAAT2 and claimed to be the cause of EAAT2
downregulation found in ALS patients. However, subsequent
studies have contradicted these findings and showed that
abnormal EAAT2 transcripts were also found in areas of the
CNS unaffected by ALS, and in normal subjects, thus ques-
tioning the proposed link between intron-retention and exon-
skipping EAAT2 mRNA variants as a cause for EAAT2 loss in
ALS pathogenesis (48, 67, 95, 97).
Similar to sporadic ALS patients, mouse models of ALS also
show a clear and consistent reduction in glutamate-transport
GLUTAMATE TRANSPORTERS IN ALS 1591

activity and EAAT2 protein levels. In mutant SOD1 mice,
several studies have shown decreased EAAT2 protein and
downregulation of glutamate-transport activity in affected
CNS areas (9, 15, 20, 23, 41, 151, 153, 155). Similar results are
found in the SOD1-G93A and H46R transgenic rats (41, 68)
(see also Fig. 2). One exception is a study from Heiman-
Patterson and colleagues (37), in which the authors showed
that EAAT2 levels in sensorimotor cortex, brainstem, and
cervical and lumbar spinal cord of G93A mice did not differ
significantly from controls, either at presymptomatic, early at
onset, or at the end stage. Although puzzling, this latter study
is interesting because these authors found retarded gel mo-
bility of EAAT2 in the brainstem, cortex, and spinal cord of
SOD1-G93A mice compared with controls. EAAT1 and
EAAT3 were unchanged in both amount and mobility. The
changes in EAAT2 mobility and distribution indicate that this
transporter could be posttranslationally altered in mice with
the SOD1 mutation. Evidence in the literature thus far has
shown no decrease of EAAT2 mRNA levels in the spinal cords
of transgenic mice, even at stages in which EAAT2 protein
could be lost (9). In addition, no quantitative change in mRNA
for EAAT1, EAAT2, or EAAT3 was found in the motor cortex
of ALS patients, including patients with a large loss of EAAT2
protein (95% decrease compared with control) and decreased
tissue glutamate transport (73% decrease compared with
control), suggesting that the dramatic abnormalities in EAAT2
expression levels may be due to translational or posttransla-
tional processes. In support of posttranslational–mediated
impairment and loss of EAAT2, several lines of evidence exist.
EAAT2 is a selective molecular target for some of the patho-
logic mechanisms occurring in ALS. Oxidative or nitrosative
stressors produce rapid inactivation of the transporter activity
(109, 139). When cultured astrocytes expressing endogenous
levels of EAAT2, MDCK cells transiently expressing EAAT2,
or Xenopus oocytes expressing EAAT2 are transfected with
ALS-causing SOD1 mutations, a marked reduction in trans-
porter activity and protein levels is seen (133, 138, 148). Ana-
lysis of chimeric transporters indicates that the EAAT2
cytosolic C-terminus domain could drive the specific degra-
dation and removal of the transporter from the plasma
membrane (148). The mechanisms of EAAT2 downregulation
in vivo in ALS are, however, not completely understood.
Several studies in the mouse model of ALS showed that
changes in the EAAT2 expression levels and glutamate-
uptake activity are found only in the ventral horn of the
affected spinal cord at a late stage of disease (9, 23, 41). An
FIG. 2. Expression levels of EAAT2 are decreased in spinal cord homogenates prepared from SOD1-H46R rat model of
ALS. Representative results from three different experiments showing Western blot analysis of spinal cord homogenates
(A–D) and hippocampus (E, F) of SOD1-H46R rats at presymptomatic stage (lane 1) and disease end-stage (lane 2). Spinal
cords and hippocampi were collected and immediately homogenized on ice (glass-Teflon homogenizer; 1,000 rpm) in 30
volumes of extraction buffer containing SDS (1%), 150 mMNaCl, 10 mMNaPi (pH 7.4), and Complete protease inhibitor mix
with EDTA (Roche). ‘‘Crude’’ extracts were incubated for 10min at room temperature, briefly sonicated, centrifuged (1,000 g,
4 min) to remove unsolubilized material, and immediately analyzed or stored at 808C. The homogenates prepared with this
protocol were termed SDS extracts (15). The rat model of ALS (SOD1-H46R line 4) was generated by Nagai and colleagues,
and the phenotype was described in (98). Spinal cord and hippocampus homogenates were collected at 120 days of age for
the presymptomatic stage and 160 days of age for the end stage. Blots were probed with affinity-purified polyclonal
antibodies against peptides of the glutamate transporter EAAT1–2, referred to by capital letters A (EAAT1) and B (EAAT2),
followed by numbers indicating the corresponding peptide of the rat transporter sequence. For this study, we used A522–541
(0.2 mg=ml), B12–26 (0.2 mg=ml), B493–508 (0.1 mg=ml), and B518–536 (0.2 mg=ml). (D) Coomassie Blue staining to show equal
total proteins content in lanes 1–2 of the spinal cord homogenates.
1592 FORAN AND TROTTI

interpretation of these studies points to a noncausal role for
EAAT2 impairment in ALS, because if downregulation of
EAAT2 is seen only at a late stage of the disease, that is, after
motor neurons have been for the most part lost, then these
changes may be more a consequence of the motor-neuron loss
and not a key to their demise. An additional clue supporting
this interpretation may be offered by the evidence that the
expression levels of EAAT2 are downregulated in purified
astrocytes in primary culture where the influences of neurons
are removed, suggesting that expression of EAAT2 is critically
dependent on the presence of neurons or at least a soluble
factor released from the neurons (122, 128).
Nevertheless, not all CNS diseases associated with neuro-
nal death display loss of EAAT2. For example, spinal mus-
cular atrophy, another motor-neuron disease characterized
by motor-neuron degeneration, has no associated loss of
EAAT2 (5, 125). If EAAT2 downregulation is a consequence
of motor-neuron loss rather then the cause, then the claim that
motor neurons could succumb to excitotoxic damage caused
by decreased glutamate-transport activity in ALS also should
be revisited. Indeed, few reports in the literature do not
support glutamate-transport deficits as a cause of neuronal
death in vivo (33, 66, 93). In a recent study, Tovar-y-Romo and
colleagues (135) attempted to verify whether blockade of
glutamate transporters could result in hyperexcitation and
loss of neurons exposed to the accumulation of extracellular
glutamate in the SOD1-G93A mouse model of ALS (135). The
expectations were that, in the disease, mouse motor neurons
and, in general, neuronal cells would be more susceptible to
excitotoxicity because of the associated oxidative environ-
ment caused by the presence of disease-causing mutant SOD1
proteins (11). Infusion by reverse microdialysis of l-trans-2,4-
pyrrolidin-dicarboxylic acid (PDC) (25 mMfor 1 h), a trans-
portable nonselective inhibitor of glutamate transporters,
directly into the hippocampus or motor cortex of SOD1-G93A
mice and SOD1–wild-type control mice caused a consistent
(about sixfold) increase in extracellular glutamate. In both
mouse models and despite the marked increase in extracel-
lular glutamate, histologic examinations showed that no
overt neuronal loss occurred in the hippocampus and motor
cortex of either SOD1–wild-type or, even more surprisingly,
SOD1-G93A ALS mice treated with PDC. These results are
quite in contrast with the hypothesis of a causal role for
glutamate-transporter impairment in ALS, because neither
increased neuronal susceptibility to excitotoxicity in the dis-
eased SOD1-G93A mice nor a correlation between elevation
in extracellular glutamate mediated by glutamate-transport
blockade and neuronal death in vivo was found. However,
some considerations should be pointed out regarding this
study: (a) the analysis of the neuronal loss was only limited to
24 h after infusion of PDC; (b) although no increased neuronal
loss was detected, the study does not indicate whether the
neurons exposed to high concentrations of glutamate were
indeed starting to become dysfunctional and still progress to
dead motor neurons if observed over a longer temporal scale;
and (c) extracellular GABA levels were not reported. An in-
crease in GABA release, which could have been induced
concurrent with the PDC-mediated increase in extracellular
glutamate, would considerably dampen the excitation of
neurons and effectively protect them from the excitotoxic
insult produced by the blockade of glutamate transporters
(28, 77, 158).
Other reports in the literature, however, showed that the
loss of the glutamate-transporter EAAT2 in the mouse and rat
models of ALS occurs also at presymptomatic and early
symptomatic stages of the disease when no overt loss of motor
neurons has occurred, suggesting perhaps that additional
mechanisms could be responsible for the selective loss of
EAAT2, independent of neuronal inputs (54, 68, 114). In the
SOD1-G93A rat model of ALS, focal loss of the EAAT2 glu-
tamate transporter in the ventral horn of the spinal cord co-
incides with gliosis, but appears before motor neuron=axon
degeneration. At end-stage disease, gliosis increases, and
EAAT2 loss in the ventral horn exceeded 90%, suggesting a
role for this transporter in the events leading to cell death in
ALS (68). In further support of this, direct manipulation of
EAAT2 expression levels has effects on both cellular and an-
imal models of ALS. Overexpression of EAAT2 was shown to
be protective in vitro (92) and to slow disease progression in
vivo (58). However, it does not prevent disease onset or death.
In one study, transgenic SOD1-G93A mice, which expressed
twice the normal levels of the EAAT2 glutamate transporter
and had twice the normal glutamate-uptake capacity in the
spinal cord, had better-preserved motor function and delayed
death of spinal motor neurons, but not delayed onset of ALS
symptoms, suggesting that EAAT2 overexpression could in-
deed afford some protection and that loss of EAAT2 may
contribute to, but does not cause, motor-neuron degeneration
in ALS (58). In this study, Guo and colleagues (50) coupled the
expression of the EAAT2 transgene to a *2-Kb fragment of
the promoter of the astrocyte-specific GFAP gene, a rather
weak promoter that becomes active at or around disease onset
(50), and thus the EAAT2 expression was progressively in-
creased only when the disease was beginning to manifest
(102). This may explain the partial protection offered by ele-
vating EAAT2 levels. In another study, Pardo and colleagues
(103) took the opposite approach and investigated whether a
further reduction in EAAT2 expression levels could accelerate
motor-neuron degeneration. They crossed the SOD1-G93A
mouse line with a mouse heterozygous for EAAT2
(EAAT2
(þ=)
). SOD1-G93A::EAAT2
(þ=)
bigenic mice exhi-
bited a significant reduction in transporter protein and
increased rate of motor decline accompanied by earlier motor-
neuron loss and reduction in survival, again underscoring a
role for EAAT2 loss in ALS (103). More recently, an elegant
study from Maragakis’s group (85) demonstrated that a sig-
nificant level of motor-neuron protection could also be
achieved by transplanting glia-restricted precursor cells
(GRPs) in the spinal cord of the SOD1-G93A rat, an interesting
application that can have therapeutic implications. What
makes this study relevant to excitotoxicity is the evidence that
neuroprotection and therapeutic efficacy of GRP cells could
have been mediated in part by the astrocytic glutamate-
transporter EAAT2 expressed in these cells.
Compounds such as the b-lactam antibiotic ceftriaxone and
GPI-1046, an immunophilin ligand, were discovered to in-
crease the levels of EAAT2 in astrocytes in culture, in spinal
cord organotypic cultures, and in vivo (52, 118). Ceftriaxone
increases EAAT2 transcription in astrocytes through the nu-
clear factor-kB (NF-kB) signaling pathway by promoting nu-
clear translocation of p65 and activation of NF-kB (84). The
mechanism of action of GPI-1046 is still not clear and must be
investigated more in detail, although the initial indication sug-
gests that immunophilins are involved in this upregulation
GLUTAMATE TRANSPORTERS IN ALS 1593

(52). Both ceftriaxone and GPI-1046 have been reported to
prolong survival and protect motor neurons in mutant SOD1
transgenic mice. Again, this may suggest that EAAT2 dys-
function contributes to disease progression and that these
drugs were neuroprotective because of their EAAT2-
enhancing effect, although no proof has been provided that
this mechanism was indeed directly responsible for the neu-
roprotection. Despite affording some degree of neuroprotec-
tion, the overall effect on the disease phenotype of these drugs
is quite unsatisfactory, casting doubts on the relevance of the
glutamate transporter–mediated excitotoxic pathway in the
degeneration of motor neurons in ALS. In this respect, one
consideration should be made in light of a recent study in
which we reported that nor-dihydroguaiaretic acid (NDGA),
an antiinflammatory compound that is also a potent gluta-
mate-uptake enhancer both in vitro and in vivo (16), failed to
increase glutamate uptake in the ALS mice because of poor
CNS bioavailability, likely caused by a disease-driven in-
creased expression of the multidrug-efflux transporter,
P-glycoprotein (16). P-glycoproteins, along with other drug-
efflux transporters, work by expelling from the cells toxins
and xenobiotics, including many potential therapeutic com-
pounds, limiting their effectiveness (87). It is therefore possi-
ble that poor bioavailability of ceftriaxone and GPI-1046 was
responsible for their modest therapeutic efficacy. The specific
mechanisms by which the expression and function of multi-
drug transporters are regulated in neurologic disorders like
ALS should also be investigated in detail and, if necessary,
should reconsider many clinical trials that have been at-
tempted in the mutant SOD1 animal model in which the in-
creased expression of multidrug transporters could have
compromised a positive outcome.
One theory to explain the depressed levels of EAAT2 ex-
pression and activity during the progression of ALS is that
aberrantly formed splice variants of the EAAT2 transcript
may prevent proper EAAT2 expression (57). Originally, these
aberrant transcripts were selectively found in ALS patients.
However, subsequent studies found these variants also in
normal controls (95) and in white matter far from the areas of
depressed EAAT2 expression (89). Another possibility is that,
although EAAT2 could be properly translated and expressed,
it loses function in ALS. This hypothesis is supported by the
evidence that a lower V
max
for EAAT2 is measured in mutant
SOD1 animal models (41). In addition, reactive oxygen spe-
cies (ROS) formed in motor neurons after glutamate-receptor
activation seem to be able to diffuse out of the motor neurons
and induce oxidation and disruption of EAAT2-mediated
glutamate uptake in neighboring astrocytes (108, 109).
Correspondingly, in a transgenic mouse model of ALS,
protein oxidation was increased in regions immediately sur-
rounding motor neurons. These results provide a mechanism
that can account for the focal loss of glial glutamate transport
seen in the disease (68) and lend support for a feed-forward
model involving reciprocal interactions between motor neu-
rons and glia, which may prove useful in understanding ALS
pathogenesis (109).
Loss of EAAT2 activity could also be mediated by a direct
or indirect effect of mutant SOD1 proteins on the transporter.
We have demonstrated that mutant SOD1 proteins linked to
familial ALS inactivate the function of EAAT2 (138). Exposure
of Xenopus oocyte cells expressing SOD1-A4V, I113T, or SOD1
wild-type as control and the glutamate-transporter EAAT2 to
the biologic oxidant hydrogen peroxide led to a rapid and
consistent inhibition of the transporter activity when either
one of the two mutant SOD1s, but not wild-type proteins,
were expressed with EAAT2. The molecular determinant(s) of
the EAAT2 inhibition resided in the cytoplasmic C-terminal
domain of the transporter. The inhibition was blocked and
even partially reversed by antioxidants such as Mn(III)TBAP,
a manganese porphyrin with SOD1-mimetic free radical–
scavenging properties, suggesting oxidation of critical resi-
dues within the C-terminal domain of EAAT2 as a possible
mechanism of inactivation. The precise site(s) of oxidation is
not yet defined, but it seems unlikely that a single amino acid
residue could be responsible for the loss of activity. However,
oxidation of the transporter mediated by mutant SOD1 pro-
teins did not alter the transport properties of EAAT2, such as
the affinity for glutamate and transport coupling coefficient,
suggesting that oxidized transport molecules are either non-
functional or form more-rigid structures that impair the
overall transport dynamics.
Interestingly, a specific disulfide reducing agent like di-
thiothreitol (DTT) was found effective in halting the inhibition
of EAAT2 but ineffective in reversing it, ruling out a major
role for disulfide bridge formation among cysteine residues as
the culprit mechanism for the inhibition. Among possible
targets of oxidation are aromatic rings of tyrosine, histidine,
and tryptophan residues, which are possibly vulnerable to
dimerization. More-extensive oxidation may also result in
modification of the thiol groups within the EAAT2 C-termi-
nus, leading to the formation of sulfenic, sulfininc, and sul-
fonic acids, which are not reducible by DTT. Also important is
the selectivity of the inhibitory action of mutant SOD1 pro-
teins toward EAAT2. The neuronal glutamate-transporter
EAAT3 does not display the same sensitivity to oxidant
stressors and mutant SOD1 proteins. Swapping the cytosolic
C-terminus domain of EAAT2 with the same domain of
EAAT3 generated a chimeric EAAT2 transporter that was
insensitive to the same inhibitory paradigm.
Searching for the molecular determinant(s) of EAAT2
sensitivity, we recently discovered that caspase-3 can cleave
EAAT2 at a unique site located in the cytosolic C-terminus of
the transporter, inactivating the transporter activity, a finding
that could link excitotoxicity and activation of caspase-3 in
astrocytes as converging mechanisms in the pathogenesis of
ALS. Interestingly, mutant SOD1 protein–mediated inhibition
of EAAT2 is also largely, although not completely (60%),
blocked by a specific inhibitor of caspase-3 and partially
prevented by disruption of the unique caspase-3 consensus
site in the cytosolic C-terminal domain on EAAT2 by site-
directed mutagenesis (15), suggesting that biologic oxidants
like H
2
O
2
in the presence of FALS-linked mutSOD1 proteins
could lead to activation of caspase-3, which in turn cleaves
within the C-terminus of EAAT2, inactivating the transporter.
What is emerging from these studies is a critical role for the
C-terminal domain of EAAT2 in regulating the transporter
activity. Whether oxidation and caspase-3 cleavage occur
concurrently or whether to be cleaved by caspase-3, the C-
terminus domain would have to be first oxidized, remains to
be elucidated. In addition, whether these processes are re-
sponsible, partially or totally, for the loss of EAAT2 in ALS
also remains to be established. In a further follow-up to these
studies, we demonstrated that a proteolytic fragment of
EAAT2 derived from caspase-3 cleavage of the EAAT2 cyto-
1594 FORAN AND TROTTI

plasmic C-terminus is SUMO1 conjugated (termed CTE-
SUMO1), progressively accumulates in the spinal cord of
SOD1-G93A mice, starting as early as the presymptomatic
stage, and this accumulation is disease specific, as it does not
occur, for instance, in the mouse model for Huntington dis-
ease (54). Moreover, we found that CTE-SUMO1 accumulates
in the nucleus of astrocytes, where it interacts with promye-
locytic leukemia protein (PML) in nuclear structures called
PML-nuclear bodies (PML-NBs) (54). Several lines of evidence
indicate that SUMOylation of polypeptides and proteins can
affect protein stability, protein–protein interactions, subcel-
lular relocalization, and transcriptional regulation (61), and
that PML-NBs interact with chromatin to regulate gene ex-
pression and DNA repair and transcription (55). This leads to
the speculation that CTE-SUMO1 may be involved in the
dysregulation of nuclear processes in astrocytes. Because as-
trocytes and neurons interact in physiology, accumulation of
CTE-SUMO1 in astrocytes may disturb or alter this relation
and ultimately lead to neuronal damage and degeneration
(Fig. 3).
Glutamate Receptor–Mediated Excitotoxicity in ALS
A chronic build-up of synaptic glutamate can cause ex-
citotoxicity. It has been shown that excitotoxic disorders are
capable of causing a dying-back neuronal phenotype similar
to that seen in ALS. Evidence of the excitotoxic aspect of ALS
is supported by the fact that the CSF of patients is excitotoxic
to cultured motor neurons compared with that from normal
controls (3, 124). In vitro, it was shown that motor neurons
are selectively vulnerable to slow excitotoxic death (94).
Motor neurons die from an influx of Ca
2þ
and mitochondria-
mediated apoptosis. The large uncontrolled influx of Ca
2þ
causes stress to the Ca
2þ
-buffering systems in the cell, in
particular the mitochondria. When the mitochondria are
overly stressed by cytosolic Ca
2þ
levels, an apoptotic cascade
can be activated concomitant with increased ROS production.
The stressed mitochondria are not capable of maintaining
proper electron activity and ROS escape into the cytoplasm
(43). Activation of glutamate (AMPA) receptors by delivery in
the mouse spinal cord of selective agonists induced motor-
neuron death. In vivo, intrathecal injection, osmotic minipump
infusion, or microdialysis perfusion of AMPA in the spinal
cord of adult rats also produced vast motor-neuron death
associated with severe motor dysfunctions (71, 99, 136). Si-
milarly, AMPA-mediated motor-neuron death can be
achieved in vivo in rat by long-term blockade of glutamate
transporters by Threo-b-hydroxyaspartate (THA) (66).
The Q=R editing site of the AMPA-receptor complex sub-
unit GluR2 is a major aspect of the AMPA-receptor ability to
block massive Ca
2þ
influx (123). The GluR2 subunit is subject
to posttranscriptional RNA editing, which results, under
normal circumstances, in a change of the glutamine residue
(Q) in the Q=R site of the AMPA-receptor subunit GluR2 to
arginine (R), a substitution that renders the AMPA-receptor
complex Ca
2þ
impermeable when activated (39, 96). How-
ever, if this editing is prevented, the resultant AMPA receptor
is permeable to Ca
2þ
. The importance of edited GluR2 in
FIG. 3. Posttranslational pro-
cessing of theglutamate trans-
porter EAAT2 in ALS.
Activated caspase-3, in mutant
SOD1-expressing astrocytes,
cleaves the glutamate trans-
porter EAAT2 at a unique site
in the cytoplasmic C-terminal
domain. Activation of caspase-
3 could be mediated by the
toxic gain-of-function of mu-
tant SOD1 in familial ALS,or it
might occur through some
other still-unidentified path-
way in sporadic ALS or the
non-SOD1 component of fa-
milial ALS (dashed-line path-
way). The cleavage inactivates
the transporter, leading to de-
creased clearance of synapti-
cally released glutamate and
persistent activation of gluta-
mate receptors (excitotoxicity),
which could contribute to the
death of motor neurons in ALS
(solid-line pathway). This pro-
teolytic cleavage also releases a C-terminal fragment of EAAT2 (CTE), which we found to be posttranslationally modified by
SUMO1 (CTE-SUMO1). SUMOylation of the CTE can occur before or after the fragment has been released from the full-length
transporter. Once released, CTE-SUMO1 accumulates in the nucleus of the astrocytes, where it associates with promyelocytic
leukemia protein (PMLs) in subnuclear structures called PML-nuclear bodies (dotted-line pathway). Within these nuclear struc-
tures, CTE-SUMO1 may disrupt their normal functions, altering the genotypic profiles of the astrocytes, indirectly contributing
to motor-neuron death or dysfunction. Both solid- and dotted-line pathways may together participate in the death processes of
motor neurons in ALS.
GLUTAMATE TRANSPORTERS IN ALS 1595

neuronal survival is indicated by the phenotype of transgenic
mice in which the RNA editing at the Q=R site is reduced. This
generates a lethal phenotype characterized by seizures and
acute neurodegeneration (21). GluR2-editing defects also
have implications in ALS, because a significant reduction in
RNA editing of GluR2 at the Q=R site occurred specifically in
motor neurons of five patients with sporadic ALS (74, 81).
In support of a crucial role for GluR2 in controlling Ca
2þ
influx through the AMPA-receptor complex, the evidence
indicates that the overexpression of a GluR2-deficient Ca
2þ
-
permeable AMPA receptor in mice leads to a late-onset motor-
neuron degenerative disorder, which is exacerbated when
coexpressed with the mutant SOD1 transgene (78, 79). To
assess the role of Ca
2þ
-permeable AMPA receptors on the
evolution of ALS pathology in vivo, Yin and colleagues (157)
examined the effects of prolonged intrathecal infusion of the
AMPA channel blocker, 1-naphthyl acetylspermine (NAS), in
SOD1-G93A transgenic rat. In wild-type animals, immuno-
reactivity for EAAT2 was particularly strong around ventral
horn motor neurons. However, a marked loss of ventral-horn
EAAT2 was observed, along with substantial motor-neuron
damage, before onset of symptoms (90–100 days) in the
SOD1-G93A rats. Compared with sham-treated SOD1-G93A
animals, 30-day NAS infusions (starting at approximately 70
days of age) markedly diminished the loss of both motor
neurons and of astrocytic EAAT2 labeling (157). Interestingly,
a recent study showed that, under normal conditions, astro-
cytes regulate the expression and subunit composition of the
AMPA receptors in motor neurons. However, under disease
conditions, this regulatory action is decreased, and the level of
GluR2-deficient AMPA receptors increases (141).
Although most attention has been focused on the AMPA
receptor, involvement of other glutamatergic receptors in ALS
has been noted. The glutamate receptor mGluR5 is implicated
in morphologic disarrangement restricted to astrocytes di-
rectly surrounding spinal motor neurons in the mutant SOD1
mouse model of ALS (114). This degenerative process of the
astrocytes manifests early at onset and becomes significant
concomitant with the loss of motor neurons and the appear-
Table 3. Some of the therapeutic trials on transgenic SOD1-G93A mice with compounds
involved in glutamate-mediated excitotoxicity
Agent (mechanism of action
with respect to glutamate
homeostasis) Dose Route
Start of
therapy
(days)
Onset
change
(%)
Survival
change Reference
Carboxyfullerene (block
excitotoxicity mediated
NMDA and AMPA receptors)
15 mg=kg=day i.p. 73 Increase Increase 40
Ceftriaxone (increase
expression levels and
activity of EAAT2)
200 mg=kg=day i.p. 42 Increase þ11% 118
Gabapentin (decrease of
glutamate release)
3% diet diet 50 þ5% 59
GPI-1046 (increase
expression levels and
activity of EAAT2)
50 mg=kg
twice a day
p.o. 150 - SOD1-
G93A low
expressors
þ12% 52
Memantine (noncompetitive
NMDA antagonist)
10 mg=kg
twice a day
s.c. 70 þ7% 150
NBQX (AMPA receptor
antagonist)
8mg=kg 5
times a week
i.p. 70 Increase þ10 143
Riluzole (decrease of
glutamate release, increase
of glutamate uptake)
50 mg=kg=day p.o. 50 þ10% 59
Topiramate (decrease of
glutamate release, block of
AMPA receptors)
50 mg=kg p.o 30  91
NDGA (increase of
glutamate transport)
1mg=day s.c. 90  16
MPEP (block of mGluR5) 30 mg=kg=day i.p. 40 Increase Increase 114
ZK 187638 (noncompetitive
antagonist of AMPA receptor)
140 mg=kg once
in 2 days
p.o. 77 Increase Increase 134
i.p., intraperitoneal; p.o., per osmosis; s.c., subcutaneous; NBQX, 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzoquinoxaline-2,3-dione; MPEP, 2-
methyl-6-(phenylethylnyl)pyridine; NDGA, nordihydroguaiaretic acid.
Table 4. Modifier genes of transgenic mutant SOD1
mice involved in Excitotoxicity
Model
Change
in
onset
Change
in
survival Reference
EAAT2
(GFAP)
SOD1-G93A þ19% 58*
EAAT2
(þ=)
SOD1-G93A 4% 103*
GluR2
(=)
SOD1-G93A 15% 15% 142*
GluR2
(ChAT)
SOD1-G93A þ20% þ14% 131*
GluR2-R607NSOD1-G93A 7% 78*
*The promoter element used to drive expression of the transgene.
1596 FORAN AND TROTTI

ance of clinical symptoms. Blocking this receptor in vivo slows
astrocytic degeneration, delays onset of ALS, and slightly
extends survival in SOD1-G93A transgenic mice. The group 1
metabotropic receptors have also been implicated in the CSF-
mediated toxicity in cultured motor neurons. Treatment with
the specific group 1 mGluR antagonist 1-aminoindan-1,5-di-
carboxylic acid selectively protects motor neurons from cell
death when treated with the CSF of ALS patients (3).
Concluding Remarks
During the course of ALS, problems in both glutamate-
receptor and glutamate-transporter systems culminate in an
excitotoxic disorder, which could adversely affect the motor
neurons. Both genetic and pharmacologic lines of evidence
suggest that the glutamatergic system is crucial to the normal
functioning of the synapses in the spinal cord, and its dysre-
gulation could play a role in the disease of the motor system
(Tables 3 and 4). Over the last 15-year period, many patho-
genic mechanisms have been proposed to take part in ALS
pathogenesis, and excitotoxicity is only one among them
(104). This multitude of factors and mechanisms, however,
indicates that not everything in ALS pathogenesis can be re-
lated to excitotoxicity, but what is emerging is that at least
some of these mechanisms are interconnected. For example,
several lines of evidence indicate that excessive stimulation
of glutamate receptors, perhaps due to impairment to the
glutamate-transport system and, in particular, of the astro-
glial transporter EAAT2, could lead to Ca
2þ
overload in mi-
tochondria, resulting in overproduction of ROS and oxidative
stress–mediated motor-neuron damage. In addition, motor
neurons could become more sensitive to glutamate-mediated
excitotoxicity in the presence of mutant SOD1 in mitochon-
dria (76).
Drugs targeted to increase EAAT2 activity, the glutamate-
transport system in general, or to block the AMPA receptors
have been shown to prevent excitotoxicity in several models
and could be potential treatments for disorders like ALS
(Table 3). However, considering the poor therapeutic efficacy
of these compounds in vivo, it is not clear whether targeting
the excitotoxic pathways in ALS could result in a therapy for
patients. In recent years, important advances have been made
in understanding basic molecular mechanisms governing the
expression and activity of glutamate transporters, their
translational and posttranslational processing, and their in-
volvement in regulating and shaping the excitatory neuro-
transmission. Considerable advances have been also made in
the field of glutamate receptors with the design of more-
specific inhibitors that can affect the different subclasses and
subtypes of receptors. These achievements are expected to
facilitate further studies on the role of individual transporter
and receptor subtypes and to develop new strategies for the
treatment of ALS and other diseases associated with mal-
functioning of glutamate transporters and dysregulation of
glutamatergic neurotransmission.
Acknowledgments
This work was supported by grants from NIH (NS044993)
and Muscular Dystrophy Association. The Weinberg Unit for
ALS research at Thomas Jefferson University is supported by
the Farber Family Foundation.
Abbreviations
ALS, Amyotrophic lateral sclerosis; AMPA, a-amino-
3-hydroxy-5-methyl-4-isoxazole propionic acid; CTE, C-
terminus of EAAT2; EAAT, excitatory amino acid transporter;
ONOO

, peroxynitrite; SOD1, superoxide dismutase 1;
SUMO, small ubiquitin modifier; ROS, reactive oxygen
species.
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Address reprint requests to:
Davide Trotti, Ph.D.
Weinberg Unit for ALS Research
Farber Institute for the Neurosciences
Thomas Jefferson University
900 Walnut Street, 4
th
floor, JHN bldg.
E-mail: davide.trotti@jefferson.edu
Date of first submission to ARS Central, January 13, 2009; date
of acceptance, January 24, 2009.
1602 FORAN AND TROTTI