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Neuropsychopharmacology: The Fifth Generation of Progress |
Amyotrophic Lateral Sclerosis, Glutamate, and Oxidative Stress
Andreas Plaitakis and P. Shashidharan
Amyotrophic lateral sclerosis (ALS) is a devastating human disease affecting
approximately one to two persons per 100,000 population each year—an incidence
similar to that of multiple sclerosis. Clinically, ALS is manifested by muscle
weakness, wasting, spasticity, and weight loss; pathologically, it is characterized
by degeneration and loss of the motor neurons in the spinal cord, brainstem,
and cerebral cortex. Death usually occurs within 2–5 years after the onset of
symptoms. While the overwhelming majority of cases are sporadic (primary or
idiopathic ALS), about 5–10% of patients have a positive family history (familial
ALS).
The
cause of primary ALS is unknown, and no effective treatment is currently available.
Recent studies, however, have shown that abnormal glutamate metabolism occurs
in patients with sporadic ALS and is thought to cause motor neuron degeneration
via neuroexcitotoxic mechanisms. Meanwhile, molecular genetic analyses have
linked the familial form of ALS with mutations of Cu/Zn superoxide dismutase
(SOD1), an enzyme that is part of the cellular antioxidant defense systems.
Because there are indications that glutamate excitotoxicity is associated with
increased oxidative stress, and that failure of antioxidant defense systems
can lead to excitotoxicity, these observations suggest common final pathways
to motor neuron degeneration. Because glutamate is central to the main theme
of this chapter, the role of this amino acid in the biology of nerve tissue
in health and disease is discussed in detail (see also A
Critical Analysic of the Neurochemical Methords for Monitoring Transmitter Dynamics
in the Brain, Exciatotry
Amino Acid Neurotransmission, Galanin:
A Neuropepptide with Important Central Nervous System Actions, and Luteinzing
Hormone-Releasing Hormone Neuronal, this volume).
GLUTAMATE
FUNCTION IN METABOLISM
Glutamate, a five-carbon
skeleton dicarboxylic amino acid, is known to play a key role in mammalian intermediary
metabolism. Glutamate is involved in the synthesis and/or catabolism of many
compounds, including amino acids, ketoacids, and peptides. Gamma-aminobutyric
acid (GABA), a major inhibitory neurotransmitter in the
mammalian central nervous system (CNS), is known
to be formed from glutamate (Fig. 1) by decarboxylation.
Glutamate can be reversibly transaminated via glutamate oxaloacetate transaminase
(GOT) with oxaloacetate to form a-ketoglutarate and aspartate
(Fig. 1). Also, it can be oxidatively deaminated
to a-ketoglutarate and ammonia via glutamate dehydrogenase
(GDH). As such, glutamate is associated directly with aerobic metabolism via
the Krebs cycle.
Glutamate
plays an essential role in ammonia homeostasis. In mammalian liver, oxidative
deamination of glutamate (via GDH) provides ammonia for urea synthesis. However,
in brain, as in other organs that do not have an active urea cycle, formation of glutamate and glutamine
via amination of a-ketoglutarate
(GDH reaction) and glutamate (glutamine synthetase reaction), respectively,
is essential for ammonia detoxification. Glutamate is an
important building block for polypeptides and accounts for over 10% of amino
acid residues present in most proteins. It is also a constituent of many biologically
active oligopeptides such as glutathione, a tripeptide present at high concentrations
intracellularly and thought to be involved in cellular mechanisms dealing with
oxidative stress (see below).
GLUTAMATE
AS EXCITATORY TRANSMITTER
Curtis
and Watkins (17) demonstrated in 1960 that
glutamate and other acidic amino acids can produce potent excitation of spinal
neurons. However, the possibility that excitatory amino acids serve as neurotransmitters
in mammalian CNS was initially discounted for a number of reasons. Glutamate
and other acidic amino acids are present at high concentrations in all cells.
These compounds show little regional variation in their distribution in mammalian
brain and are also known to be involved in many aspects of intermediary metabolism.
These characteristics are in contrast to those of other biologically active
molecules
(i.e., dopamine, norepinephrine, and acetylcholine) considered as classic neurotransmitters.
In spite of these features, evidence has mounted over the past decade in favor
of the neurotransmitter role. In fact, glutamate has been accepted as having
satisfied the main criteria required for classification as an excitatory transmitter
in mammalian CNS. (see The
Psychopharmacology of Sexual Behavior, this volume).
Glutamate Compartmentation
A
key feature that may allow glutamate to perform its many functions in nerve
tissue is compartmentation of its distribution into distinct pools (21).
The largest of these pools (about 50% of total) is related to the metabolism
of neurons and is known as the metabolic pool. A smaller neuronal pool
(20–30% of the total) is releasable from the nerve endings during neurotransmission
and is thought to represent the glutamate neurotransmitter pool. A separate
pool (10–30% of the total) is contained in glial cells and is believed to serve
the recycling of transmitter glutamate (glial pool). The smallest glutamate
pool (5% of the total) is believed to be involved in the synthesis of GABA (GABA
precursor pool).
Glutamate Transport
Transmitter
glutamate is stored presynaptically in specific nerve endings (glutamatergic)
where it can be released by a calcium-dependent mechanism (21). The extracellular
concentration of glutamate is very low (1-3 µM) except during excitatory impulses
when the concentration in the synaptic cleft can reach 1-2 mM (14). The synaptic action of the amino acid is believed
to be terminated by rapid removal from the synaptic cleft via a high-affinity
uptake system, which is sodium-dependent and which does
not discriminate between glutamate or aspartate. This system is present both
in the surrounding astrocytes and in the nerve terminals, with the glial uptake
thought to be particularly efficient.
Glutamate uptake
is accomplished via high affinity transport proteins (glutamate transporters
or carriers) which are present on the plasma membrane of both neurons and glial
cells. Initial efforts led to the cloning of three mammalian sodium-dependent
excitatory amino acid transporters (EAAT). Two of these, originally designated
GLAST (84) (present name: EAAT1) and GLT-1 (58)(present name: EAAT2), are expressed in brain
and are localized in glial cells (32,58,84). However, more recent studies (40) revealed
that GLT-1 (EAAT2) is also localized in neurons. The third one, originally designated EAAC1 (30) present name: EAAT3), is expressed both in brain (localized
in neurons) and in peripheral tissues (30,76,77). We (75-77)
and others (3)have cloned the corresponding human glutamate
transporters. Recently, two additional glutamate transporters, designated EAAT4
(20) and EAAT5 (4), have been cloned: EAAT4 is predominantly expressed in the cerebellum
and has the properties of a ligand-gated chloride channel (20), whereas EAAT5 is expressed predominantly in the retina
(4).
With respect to the regional
localization of the glutamate transporter proteins, immunocytochemical studies
(22,23,32,78) using antibodies against synthetic peptides corresponding
to specific regions of the deduced amino acid sequences for these proteins revealed
that EAAT1 (GLAST) is preferentially expressed in the molecular layer of the
cerebellum, whereas EAAT2 (GLT-1) is widely expressed in mammalian CNS. In the
spinal cord, EAAT2 is expressed both in the ventral and dorsal horns, with the
latter expression being particularly prominent. EAAT3 is also widely expressed
in mammalian CNS where is localized in neuronal somata, dendrites and fine-caliber
fibers. EAAT4 is predominantly expressed
in the cerebellum where is localized in the Purkinje cells; lower levels of
expression are found in the forebrain (23)
Glutamine/Glutamate
Cycle
Synaptic glutamate, removed by uptake into the
surrounding glial cells, is believed to be recycled via the glutamine/glutamate
cycle (21). The first step of this cycle is amination of glutamate to
glutamine via the action of glutamine synthetase, an enzyme of exclusive glial
localization. Glutamine, thus formed, readily crosses cell membranes and is
transported back to the nerve terminals where it can be converted to transmitter
glutamate via the action of the neuronal enzyme glutaminase. In addition, glutamate
taken up by glial cells may be oxidatively deaminated by GDH or transaminated
by GOT to a-ketoglutarate,
which can then enter the Krebs cycle and be oxidized to CO2 and H2O, or be recycled, serving as a precursor of transmitter
glutamate.
Recycling
of glutamate at the glutamatergic synapses seems to be essential for preventing
depletion of the transmitter because the brain seems to have a rather limited
capacity to synthesize glutamate de novo from glucose. Synthesis of glutamate
from glucose removes a-ketoglutarate
from the Krebs cycle which, if not replenished, will eventually result in the
failure of the cycle. On the other hand, replenishment of five-carbon skeleton
substrates of the Krebs cycle requires fixation of CO2 on pyruvate (Fig. 1)
via either the pyruvate carboxylase or the malic enzyme reaction resulting in
the formation of oxaloacetate and malate, respectively (anaplerotic reactions).
These anaplerotic reactions are estimated to be only one-tenth as active in
the brain as in the liver (89). However, enhanced
CO2 fixation has been found in brain under conditions
of increased ammonia load and attributed to de novo production of a-ketoglutarate, required for the synthesis of
glutamate and glutamine as a means of ammonia
detoxification.
Glutamate
Receptors
Postsynaptic transduction of excitatory transmission is mediated
by several classes of glutamate receptors . These include the NMDA (N-methyl-D-aspartate), the AMPA [amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic
acid]/kainate, and the metabotropic receptors. These receptors not only exhibit
different ligand specificity, but they also differ in such features as duration
and speed of postsynaptic transduction, desensitization, and other characteristics.
The NMDA and the AMPA/kainate receptors are called ionotropic
receptors since they
are coupled to ion channels, whereas
the metabotropic receptors mediate their action through G proteins.
The NMDA receptor is highly permeable to Ca2+ and can be blocked by magnesium in a voltage-dependent
manner. NMDA, quinolinic and ibotenic acid are selective agonists, whereas ketamide
and phencyclidine are selective antagonists at this receptor. Glycine has been
shown to potentiate excitatory transmission by acting on a strychnine-insensitive
allosteric site of the NMDA receptor (37) (Fig. 2). Transduction
through the NMDA receptor produces slow but sustained physiologic responses.
In addition to its role in glutamatergic transmission, the NMDA receptor is
involved in neural development and activity-dependent synaptic plasticity. Long-term
potentiation is a long-lasting increase in synaptic sensitivity that can be
induced by high-frequency stimulation and that has been implicated in memory
and learning processes. Molecular biological techniques have led to the cloning
of two main subunits, designated NR1 and NR2, of the NMDA receptor in the rat
(47). Subunit NR1 is ubiquitous and consists of seven isoforms
generated by alternative mRNA splicing. Subunit NR2 is regionally localized
and consists of four subtypes encoded by different genes.
The
AMPA/kainate receptors seem to mediate predominantly fast excitatory synaptic
transmission . Molecular cloning of these receptors has led to the realization
that they exist physiologically at heteromeric combinations of multiple subunits
(GluR1-GluR7; KA1-Ka2) with different ligand specificity (47). Depending on the particular subunit composition, the
AMPA/kainate receptor may or may not be permeable to Ca2+ ions (47). CNQX
and 2,3-benzodiazepine GYKI 52466 are known as nonselective and selective antagonists at the
AMPA/kainate receptor, respectively. In contrast
to the above receptors which are linked to ion channels, the metabotropic receptor
mediates its action through G proteins. Activation of this receptor stimulates
inositol 1,4,5-triphosphate (IP3) metabolism and is associated with mobilization
of Ca2+ from intracellular
stores. At the molecular biological level, the metabotropic receptor consists
of at least eight subtypes (mGluR1 through mGluR8) (47,51). trans-(±)-ACPD [trans-(±)-1-amino-1,3-cyclopetane-dicarboxylic
acid] is shown to be a selective agonist, and quisqualate a nonselective agonist,
at the glutamate metabotropic receptor.
(see Exciatotry
Amino Acid Neurotransmission, this volume, for additional details)
The Neuroexcitotoxicity
Concept
Over four decades
ago Lucas and Newhouse (35) observed
that the systemic administration of monosodium glutamate to experimental animals
resulted in degeneration of retinal ganglion cells. Olney et al. (50) subsequently
showed that excitatory amino acids given systemically to immature animals can
cause neuronal degeneration in areas of the brain that
lack an intact blood–brain barrier, such as the
hypothalamus. They further noted that the neurotoxicity of acidic amino acids
correlates with their ability to excite nerve cells and coined the term "neuroexcitotoxicity."
These observations
aroused considerable interest
because the pattern of neuronal degeneration induced by the neuroexcitotoxic
compounds are similar to that found
in some disorders with system atrophy, such as in Huntington's disease. Whether the pathogenesis of these
disorders relates to excitotoxic mechanisms, as originally suggested, remains
unclear. The recent cloning of
the gene responsible for Huntington's disease opens new avenues for testing
this hypothesis and elucidating the primary pathogenetic process in this disease.
Glutamate dysfunction has also been suggested in a variety of neuropsychiatric
disorders, including schizophrenia (see Mood
Disorders Linked to the Reproductive Cycle in Woman, this volume).
Neuroexcitotoxicity
in Metabolic Encephalopathies
Around the time when the potential role of excitotoxicity
in neurodegeneration was first suggested, evidence implicating altered glutamatergic
mechanisms in nerve cell death induced by various metabolic insults began to
accumulate. Initial studies on experimental thiamine deficiency encephalopathy
revealed altered glutamate uptake and metabolism in the brains of deficient
animals (59). Additional studies (31) have shown attenuation of brain lesions with
the use of NMDA receptor antagonists. Also, metabolic alterations induced in
the brain by the selective neurotoxin 3-acetylpyridine led to the finding that
the glutamate-metabolizing enzyme glutamate dehydrogenase is reduced in patients
with neurodegenerative disorders characterized by multiple system degeneration
(60). The systemic metabolism
of glutamate was
found to be altered in these patients (60).
Glutamatergic excitotoxic mechanisms have also been implicated in the nerve
cell death that occurs in hypoxia and in hypoglycemia.
As indicated above, dysregulation of glutamate
metabolism occurs in patients with multisystemic neurologic disorders associated
with decreased glutamate dehydrogenase activity (60). Because motor neuron involvement
is often encountered
in such patients, it was suggested that innervation of anterior horn cells by the glutamatergic
corticospinal fibers and interneurons renders these
cells susceptible to the neurodegenerative process. The possibility was further
raised (61) that abnormalities of glutamate metabolism
may also occur in patients with primary ALS and be responsible for its neurodegeneration.
Plaitakis and Caroscio (61) accordingly
measured amino acid levels in the fasting plasma from 22 patients with motor
neuron disease (MND) (15 males, 7 females), 19 of whom had typical ALS (upper
and lower motor neuron deficits). Results revealed that plasma concentrations
of glutamate were selectively elevated (40.8 ± 12.7 mM; p < 0.001) in the ALS patients when compared
to age-matched healthy controls (21.3 ± 7.9 mM) and to patients with other types of neurodegenerative
or neuromuscular disorders (disease controls). Oral loadings with monosodium
glutamate produced excessive elevations in plasma glutamate associated with
proportional increases in plasma aspartate levels (61). Defective glutamate transport across cellular or mitochondrial
membranes (glutamate-OH translocator linked to the oxidative deamination pathway)
was thought to be responsible for the abnormal glutamate clearance. On the other
hand, the rise in plasma aspartate was consistent with an intact transamination
pathway, including a normally functioning glutamate/aspartate translocator (61,62).
These metabolic abnormalities were similar to those observed in patients with
GDH deficiency, although the activity of this enzyme was normal in leukocytes
of ALS patients (61).
More
extensive investigations were undertaken involving 88 patients with MND (64) . Of these, 62 had typical ALS, 23 had progressive
bulbar palsy (PBP), and 3 had progressive muscular atrophy (PMA). As compared
to control values, glutamate levels were increased by about 80% (p <
0.0001) in patients with typical ALS and by about 30% (p < 0.005)
in those with PBP. No changes were found for PMA patients, but the number of
such patients studied was very small. Nevertheless, it appeared
that dysregulation of glutamate
metabolism occurs primarily in patients with typical ALS.
Perry
et al. (54) studied 28 patients with various types of motor
neuron disease. Of these, 16 had typical ALS, four suffered from disease limited
to lower motor neurons (i.e., PMA) (C. Krieger, personal communication),
seven had PBP, and one had familial ALS. Glutamate values in normal controls
were 25 ± 12 mM (N
= 48), and those in the mixed group of 28 MND patients were 33 ± 19 mM (p < 0.05). Perry et al. (54) attributed these differences to patients' older ages,
although another study by the same authors, which was reported concurrently and evaluated 98 controls (55), showed no effect of aging on plasma glutamate
levels. If the data are analyzed according to disease type (66), patients with typical ALS have plasma glutamate
levels of 38.2 ± 18.7 mM (N
= 16), which are close to those obtained in our laboratory (61) as described above.
Blin
et al. (7) studied 18 patients (12 males, 6 females) with primary ALS.
They found significant glutamate elevations in the plasma of the ALS patients
(168.3 ± 57.2 mM; p < 0.01) as compared to healthy
controls (57.1 ± 31.7 mM; N = 16). Also,
Iwasaki et al. (29) determined amino acid levels in the plasma of 10
ALS patients (6 males, 4 females) and compared them to 10 normal controls. Glutamate
levels were significantly greater in the ALS (163.9 ± 117.3 mM; p < 0.001) than in the control group (34.1
± 11.3 mM). Plasma aspartate and glycine levels showed
lesser increases. Recently, Babu et al. (5)
reported that blood glutamate levels were significantly higher in ALS patients
than in controls. However,
Shaw, et al. (81) found no significant differences in the fasting plasma
levels of 22 amino acids between 37 patients with MND and 35 neurological controls.
Several investigators have measured amino acid levels in the cerebrospinal fluid
(CSF) of ALS patients and have obtained seemingly
conflicting results (54, 69, 70,
81). It is now well established
that levels of glutamate in the CSF are extremely low (~0.3 mM), whereas those of glutamine are as high as those present
in plasma (~660 mM). Therefore,
measurement of CSF glutamate is much more difficult than that of plasma. Because
glial cells have a tremendous capacity to eliminate transmitter glutamate, it
remains uncertain whether any glutamate measured in CSF reflects that present
in the CNS extracellular space. It has been our experience (unpublished data)
that, in controls, CSF glutamate is present at trace levels or is nondetectable.
Rothstein et al. (70), used a very sensitive analytical method to measure amino
acids in the CSF
and reported control glutamate
values which were
about 10-fold lower than those
previously reported by the same authors utilizing different methodology
(69). Both of these studies (69, 70) showed that glutamate
and aspartate concentrations were significantly elevated in the CSF of ALS patients
when compared to those of controls. Perry et al. (54), who have reported control CSF amino acid values similar
to those of Rothstein et al. (70),
did not find any differences in CSF glutamate levels between ALS patients and
controls. However, Perry et al., (54) did not specify in their report the clinical
syndromes of 17 ALS patients on whom CSF measurements were made. Recently, Shaw et al., (81)
reported that CSF glutamate levels were increased in a subgroup of patients
with MND. Hence, it is reasonable to conclude that the
conflicting results on CSF glutamate in ALS may be attributable to the great technical difficulties
involved in determining these levels and/or to the selection of patients.
Central Nervous System:
Reduced Intracellular Glutamate Pools
In
contrast to the elevated plasma and CSF levels, the concentration of glutamate
was significantly decreased in all brain and spinal cord areas studied of ALS
patients (36, 53,
62, 87). In absolute terms,
the decrease in glutamate was the same in all brain areas studied (about 2 mM/gram wet tissue) (53, 62). However, there were
greater proportional decreases in the
spinal cord due to its normally lower glutamate content. Changes in glutamate
levels were selective because other amino acids were not significantly altered
except for aspartate, which was reduced in the cervical and the lumbar spinal
cord of ALS patients (36, 62, 87).
Reductions
in glutamate levels did not correlate with regional degenerative changes occurring
in ALS. Thus, decreased glutamate content was found not only in pathologically
affected areas such as the spinal cord, brainstem, and motor cortex (36, 53,
62, 87), but also in the
basal ganglia, hippocampus, occipital cortex, and cerebellum (53, 62)—regions that are spared in the degenerative process. Also within
the spinal cord, glutamate and aspartate decreases were observed even in the
dorsal horns (36), which are pathologically spared in ALS. Since
almost all glutamate measured in nerve tissue is intracellular (the extracellular
levels are extremely small), these data are consistent with depletion of the
intracellular CNS glutamate pools.
The above data are thought to suggest that the distribution of glutamate is altered between its intracellular and extracellular pools
in ALS (61). Impaired
glutamate transport across mitochondrial and/or cytoplasmic membranes may be implicated (62-63).
Defective mitochondrial transport (glutamate-OH translocator) could affect the
oxidation of glutamate by these organelles, whereas defective cytoplasmic transport,
including impaired glial or neuronal high affinity uptake, as suggested by Plaitakis (63), could impair
the detoxification of synaptic glutamate.
Rothstein
et al. (71) have accordingly measured the high-affinity
glutamate uptake in nerve tissue of ALS patients obtained at autopsy. They found
decreased accumulation of [14C]glutamate by synaptosomes isolated from spinal cord, motor cortex, and
somatosensory cortex . However, synaptosomes from visual cortex, hippocampus
and striatum showed normal glutamate uptake. Kinetic analysis revealed decreased
Vmax
but normal Km. These findings are indicative of a decreased number of uptake sites
but normal transport affinity. In addition, Shaw et al. (79), using quantitative autoradiography, showed that the
specific binding of [3H]D-aspartate was
decreased in the intermediate grey matter and in the substantia gelatinosa of
the lumbar cord of ALS patients. Patients with progressive muscular atrophy
(PMA) showed lesser changes. The authors suggested
that these changes may be due to a loss of glutamatergic terminals of the corticospinal tract, which occurs primarily in ALS;
involvement of this track is less conspicuous in PMA. Although is unclear whether these uptake changes are
primary or secondary to the disease process, studies
utilizing organotypic cultures of rat spinal cord have shown that chronic treatment
with glutamate uptake inhibitors leads to loss of motor neurons (72).
Following the cloning of cDNAs encoding for three excitatory amino acid
transporters, Rothstein et al. (73)
used antibodies against synthetic oligopeptides corresponding to C terminal
region of each of these transporters to study CNS tissue from ALS patients.
Western blot analysis of tissue homogenates revealed a 30-95% decrease in EAAT2
immunoreactivity in 60-70% of patients with sporadic ALS. These changes were
found in pathologically affected areas (spinal cord and motor cortex), but not
in other CNS regions. The decrease in EAAT2 was selective since EAAT1 and EAAT3
immunoreactivity were not significantly altered. Patients with SOD1 mutations
showed no significant decreases in EAAT2 immunoreactivity.
Rothstein’s group (9) originally reported
that Northern blot analyses of brain and spinal
cord tissue from ALS patients lacking EAAT2 showed no significant changes in the levels or the size of the mRNA encoding
this protein. However, additional studies by the same group of investigators
(34), using primarily the Reverse Transcriptase-Polymerase Chain Reaction
(RT-PCR) method, led to the amplification of mRNAs that were distinct
from the previously described human brain cDNAs
specific for EAAT2 (3, 77). Two
new mRNAs, designated “aberrant mRNA
species” were detected in ALS patients: one that retains the 7th intron of the EAAT2 gene resulting in a truncated
protein and another with exon 9-skipping. Surprisingly, these were limited
to the pathologically affected
CNS regions (34). The authors speculated that the “aberrant mRNAs” were
the cause of the decreased EAAT2 in ALS, a contention supported by the possibility that the abnormal mRNAs could down regulate EAAT2
protein (34). However, Nagai et al. (46), who also used RT-PCR to study EAAT2 mRNA in ALS, have recently reported that such truncated transcripts
are not disease-specific. In contrast to
the data of Rothstein’s group (34),
Nagai et al. (47) found truncated transcripts containing intronic sequences
at the 3’ end of exon 7 and exon-8 skipping, both in ALS patients and in controls.
Nagai et al. (47) attributed the presence
of these truncated transcripts to alternative splicing.
Changes in N-Acetylaspartate
and N-Acetylaspartylglutamate
In addition to glutamate, N-acetylaspartate
(NAA) is also known to be present in high concentrations in the nerve tissue.
In many regions of the mammalian CNS, NAA levels are second only to glutamate.
As with glutamate, almost all NAA content is limited to the intracellular and,
probably, neuronal compartment. N-acetylaspartylglutamate (NAAG), a dipeptide
that can excite neurons, is also present at rather high concentrations intraneuronally.
The contents of NAA and NAAG in human CNS exhibit a reciprocal distribution
pattern consistent with rostrocaudal gradient of decreasing NAA and increasing
NAAG concentrations (65). Because NAAG is thought to derive from NAA, this reciprocal
distribution may reflect different regional turnover rates with the higher spinal
cord NAAG, probably resulting from a high rate of conversion of NAA to NAAG.
Measurement
of these compounds in postmortem brain and spinal cord tissue from ALS patients
showed that both NAA and NAAG were significantly reduced in the cervical spinal
cord of these patients by 40% and 48% (p < 0.001), respectively (16, 65, 87).
In contrast, NAA and NAAG levels were not altered in postmortem frontal and
cerebellar cortex of ALS patients (65).
However, Pioro et al. (56)) using 1H-magnetic resonance spectroscopy detected decreased
NAA levels in the cerebrum of ALS patients in vivo. Similar findings
have been reported recently by Block et al. (8), who also used proton magnetic resonance spectroscopy
to show that NAA levels are decreased in the motor cortex of ALS patients
More recently, Pioro
( 57) was able to detect glutamate and glutamine in vivo using short
time (TE) proton magnetic resonance spectroscopy and spectroscopic imaging.
He used creatine/phosphocreatine (Cr) as a reference point since these compounds
(in contrast to neuron-specific NA groups) are distributed homogeneously throughout the brain.
He reported preliminary data showing that, although the NA/Cr values were decreased
in sensorimotor cortex and brain stem, Glu+Gln/Cr were increased in the medulla
of these patients. The author could not determine whether elevation of Glu+Gln
precedes or follows neuronal degeneration in these patients.
Glutamate
Receptors in ALS
We (61-63) and others (69-71) have hypothesized that the abnormality in glutamate
metabolism is associated with altered presynaptic glutamatergic mechanisms leading
to an abnormally enhanced excitatory transmission mediated by glutamate receptors
and selective degeneration of postsynaptic motor neurons. Disappearance of neurons
that bear glutamate receptors is expected to cause a decrease in the density
of these receptors. Allaoua et al. (1) have accordingly measured the
density of glutamate receptors in the spinal cord of ALS patients and reported
that the NMDA receptor was decreased in these patients. Virgo and de Belleroche
(88)
also found that mRNA levels for the
NR-1 subunit of the NMDA receptor were significantly decreased (by 50%) in the
ventral spinal cord region, but not in the dorsal region. However, the mRNA levels for the NR-2 subunit were decreased both
in the dorsal horns (55%) and ventral horns (78%) of ALS patients (74).
Shaw et al. (80) studied the binding of [3H]MK-801, a selective
agonist at the NMDA site. Results showed
that [3H]MK-801 binding was decreased in
the spinal ventral horns of ALS patients. In contrast, this binding was
increased in the intermediate
spinal gray matter and deeper layers of the motor cortex. Gredal et al. (25) also found that the increased
[3H]MK-801 binding in several neocortical regions. This was associated
with an enhanced binding affinity of the ligand for the receptor
In contrast to the changes of the NMDA receptor in ALS found by Allaoua et al. (1), the AMPA/kainate receptors
were not altered (1). Morrison et al. (42) recently used SOD1 transgenic mice to study glutamate
receptor changes. They reported that GluR2-immunoreactivity was not altered
in these animals, making it unlikely that the motor neuron loss in this animal
model is mediated via the AMPA/kainate receptor.
PATHOPHYSIOLOGY
OF GLUTAMATE ALTERATIONS IN ALS
A Diffuse Metabolic
Defect?
As indicated above, there are solid data showing
that the glutamate content of brain and spinal cord is selectively decreased
in ALS patients. In absolute terms, the decrease in glutamate levels is the
same in all brain areas studied (about 2 mM/gram wet tissue). These changes do not correlate with
regional degenerative changes, since they occur both in pathologically affected
areas (spinal cord, brainstem, and motor cortex) and in CNS regions that are spared in the degenerative process (basal ganglia,
hippocampus, occipital cortex, and cerebellum). In contrast, changes in the
neuronal markers NAA and NAAG were limited to histologically affected CNS regions.
These widespread decreases of nerve tissue glutamate
cannot be attributed to neuronal loss since they occur in CNS regions that are histologically normal and maintain a normal content of
the neuronal marker NAA.
The magnitude of
glutamate decrease (approaching 50% in some CNS regions) is consistent with
an involvement of the metabolic pool
of the amino acid. This is the largest of all glutamate pools (estimated to
be 50% of total). In contrast, changes limited to the neurotransmitter glutamate
pool, resulting from degeneration of the glutamatergic nerve terminals, are
not sufficient to account for these decreases. As mentioned above, Pioro (57) reported preliminary data based on proton magnetic
resonance spectroscopy that showed that Glu+Gln/Cr
are increased in the medulla of living ALS patients. These interesting data,
if confirmed by additional studies, suggest that dysregulation of glutamate
metabolism in brain in vivo is a dynamic process. It remains to be seen whether CNS glutamate increases during the active phase of neurodegeneration
and whether this is followed by depletion of the amino acid at the end stages
of the disease (as detected in post mortem tissues).
The CNS glutamate
alterations, taken together with the systemic glutamate abnormalities, suggest
that a generalized abnormality in glutamate metabolism occurs in ALS (61-63). To this date, however, the nature of this metabolic
defect remains unclear. In this regard, observations made by Rothstein’s group are of great interest.
The changes described by these investigators (2, 34, 71-73), involving the high
affinity glutamate uptake, the EAAT2 protein immunoreactivity and the EAAT2
mRNA species were all limited to histologically affected CNS regions. In contrast,
decreased glutamate content occurs throughout the CNS (see below). Meanwhile,
detection of mutations involving the Zn/Mn SOD in familial ALS cases (see below),
a metabolic enzyme showing widespread distribution in human tissues, has provided
direct evidence that diffuse metabolic defects are capable of damaging motor
neurons selectively.
A Glutamate Transport
Defect?
Because almost all glutamate measured in nerve
tissue is intracellular and the glutamate present
in the plasma and CSF may reflect the extracellular
concentrations, the data described above are consistent with an altered distribution of the amino acid between its intracellular and extracellular pools (62). Plaitakis et al
(63) further suggested that a defect in the transport of glutamate or an increased
release of intracellular pools to extracellular space could be operational.
The finding of decreased accumulation of [14C]glutamate by spinal cord and brain synaptosomes
of ALS patients, as reported by Rothstein
et al.(71 ), is consistent
with this postulated transport defect. Decreased glutamate
transport is expected to disrupt the recycling of this transmitter with resultant
depletion of its intracellular stores (decreased nerve tissue glutamate content)
and increased synaptic (extracellular) levels. However, a careful consideration of the available data
reveals that a correlation between regional changes in glutamate content and
in the high-affinity uptake is lacking. Thus, in the striatum and visual cortex,
areas showing decreased glutamate levels (53), synaptosomal glutamate uptake was found to be normal (71).
Hence, defective uptake cannot adequately account for the widespread depletion
of CNS glutamate content in ALS.
Because experimental
lesioning of glutamatergic pathways is known to lead to decreased glutamate
uptake, it is possible that loss of
uptake, as described
by Rothstein et al. (71), reflects
disappearance of glutamatergic nerve endings in ALS. Shaw et al. (79),
who studied the spinal cord of patients with ALS and PMA, reported that the decreased
[3H]-aspartate binding in these disorders correlates with the loss of glutamatergic terminals of the
corticospinal tract. Observations on two studies of mouse models of ALS are consistent with this possibility. In the Mnd mouse, a genetic mutant used
as a model for adult-onset MND disease (6), decreased glutamate uptake was found in CNS regions analogous to those
of ALS patients (spinal cord and motor cortex but not in the striatum). These
uptake changes did not precede the onset of the neurologic abnormalities but,
instead, they occurred after the development of the neuropathological changes.
As with the human data, the high-affinity uptake of other neurotransmitters
was not altered (6).
Hence, a primary genetic defect affecting the glutamate/aspartate transport
system seems unlikely. Similar data have been obtained in mice expressing the
dominant mutation of human copper/zinc superoxide dismutase (SOD1). In this
animal, a model for familial ALS caused by a
specific genetic defect, decreased glutamate uptake in the spinal cord occurs
late in the course of their disease (13), probably reflecting loss
of motor neurons. Alternatively, oxidative damage to cellular membrane components,
such as EAAT2, could be responsible
for the decreased glutamate uptake. However, the EAAT2 was found to be normal
in patients with the SOD1 mutation (73).
A
Primary EAAT2 (GLT-1) Transporter Defect?
As described above, Rothstein et al. (73) reported that Western blot analysis of postmortem tissues revealed that EAAT2 immunoreactivity was significantly
decreased in the motor cortex and spinal cord of ALS patients. However, recent immuno-cytochemical studies
by Fray et al. (22) revealed that, although EAAT2 immunoreactivity was decreased in the spinal
cord, this transporter
was increased in the middle laminae
of the motor cortex. The authors suggested that glutamate pathology in MND may
be a more complex phenomenon than previously thought (22). It is of interest that changes
in EAAT2 immunoreactivity, as reported by Fray et al. (22 ), are similar to those
of the NMDA receptor (25,80)
and may imply that parallel alterations in synaptic elements occur in the CNS
of ALS patients.
Although decreases in EAAT2 immunoreactivity, as described by Rothstein et al. (73), appeared to be marked for
some sporadic ALS cases, the implications of these findings remain uncertain.
The fact that the EAAT2 alterations are limited to the pathologically affected
CNS regions raises the possibility
that they may represent a consequence of glutamatergic nerve terminal degeneration.
There is already ample experimental evidence showing that lesioning of glutamatergic
pathways decreases the expression of EAAT2 protein by glial cells. Levy et al.,
(33)
demonstrated that cortical lesions capable of decreasing striatal glutamate
uptake lead to reduced expression of the EAAT2 (GLT-1). Gegelashvili et al. (24) described that neuronal
soluble factors regulate the expression of this transporter by cultured astroglia.
Hence, disappearance of nerve terminals, as occurring in ALS, could eliminate the chemical signal that induces
expression of this transporter by the surrounding astrocytes.
Although Northern blot analyses of ALS tissues by Rothstein’s group (9) revealed no significant changes in the levels
or in the size of the mRNA for EAAT2, additional studies by
the same group of investigators (34) revealed the presence
of EAAT2 mRNAs
that were distinct from the previously described human brain cDNAs specific
for EAAT2 (3, 77). These were designated “aberrant mRNA species” and thought to encode truncated proteins. However, previous studies by these investigators (73) using
Western blots failed to show the presence of truncated EAAT2 proteins in ALS tissues. Nagai et al. (46)
found truncated transcripts (mRNAs) both in ALS patients
and controls and attributed these changes to alternative splicing of GLT-1 mRNA.
These authors concluded that these mRNAs are the result of physiological processes
and, as such, they do not play a pathogenetic role in ALS.
The possibility of a primary genetic defect involving the EAAT2 is not supported by recent data
obtained by Aoki et al. (2), who performed structural analyses of the gene encoding
EAAT2. These authors used genomic DNA isolated from ALS patients on whom the
Rothstein’s group had detected abnormalities both in EAAT2 protein and mRNA
(34,73). Results revealed that these patients had no structural abnormalities of the gene encoding
EAAT2. Hence,
the mechanism by which the “aberrant EAAT2 mRNAs” are generated [if not through
the physiological process of alternative splicing, as suggested by Nagai et
al. (46)] remains a mystery. In addition, data in EAAT2 gene null mice do not
support a primary role for this transporter in motor neuron degeneration. Tanaka
et al. (85), who generated mice lacking EAAT2, reported that the clinical manifestation of these animals is generalized seizures rather
than ALS. It is
known from clinical observations that epilepsy is not a feature of ALS.
In
conclusion, the new data generated by Rothstein and his co-workers on EAAT2
are quite exciting and may lead to a better understanding of ALS. However, more
extensive studies are needed to test whether the abnormalities described by
these investigators are causally related to this disorder or represent consequences
(epiphenomena) of the neurodegenerative process.
A
Primary Membrane Abnormality?
An alternative possibility
that could account for the altered glutamate distribution in nerve tissue is
an increased release of glutamate from the nerve terminals and/or increased
leakage of the amino acid through a defective cell membrane. This is expected
to lead to depletion of the intracellular stores and to increased extracellular
glutamate levels. The finding that, in addition to glutamate, other compounds
with a high intracellular/extracellular gradient, such
as aspartate, NAA and NAAG, are also reduced in nerve tissue (16, 62, 87) and increased in the CSF (69) is consistent with this possibility. NAA is an anion
that may contribute substantially to intraneuronal osmotic pressure. Hence,
reduction in the nerve tissue levels of NAA is indicative of a diffuse membrane
abnormality that may permit compounds with high intracellular
concentrations to leak out of the cell (62).
This is expected to increase the workload of the various membrane pumps responsible
for transporting these substances against a concentration gradient. Ultimately,
failure of these systems may lead to an accumulation of toxic amounts of glutamate
at the synapses and degeneration of the postsynaptic motor neurons. Detection
of mutations affecting the Cu/Zn superoxide dismutase in familial ALS (68), a defect capable
of affecting membrane permeability through lipid peroxidation (see below), is
consistent with this possibility. Recent studies by Block et al. (8) using proton magnetic resonance spectroscopy to evaluate
the primary motor cortex of ALS patients revealed alterations in the inositol
and choline, two compounds associated with plasma membrane metabolism.
A Problem of Energy
Metabolism?
Impaired energy
metabolism or increased oxidative stress (see below) could render the nerve
cells incapable of maintaining their high intracellular/extracellular gradients
for many biologically important compounds. Decreased glucose utilization has
been shown by the use of the PET technology in ALS patients (18). Defective glial uptake or metabolism could also lead
to decreased detoxification of transmitter glutamate and impaired recycling
of the amino acid at the nerve terminals. Recently, Wiedemann et al. (90) studied mitochondrial function in skeletal muscle
homogenates of 14 patients with sporadic ALS. They reported that mitochondrial
oxidative phosphorylation and the specific activity of NADH:CoQ oxidoreductase
were significantly decreased in the ALS patients as compared to 28 age matched
controls. The authors considered unlikely that these abnormalities are due to
neurogenic atrophy, since they were not present in muscle homogenates of patients
with spinal muscular atrophy.
MUTATIONS
OF Cu/Zn SUPEROXIDE DISMUTASE IN FAMILIAL ALS
The use of the "reverse genetic approach"
has led to a major breakthrough in elucidating the primary genetic abnormality
of a subgroup of familial ALS cases. The initial step was the linkage of dominant
ALS to human chromosome 21q22.1, a region containing the gene for the Cu/Zn superoxide
dismutase (SOD1). A tight linkage was found between the disease in several ALS
families and the SOD1 gene, while direct sequencing of this gene revealed the
presence of 11-point mutations in 13 families (68). Further investigations
revealed that over 50 missense mutations occur in patients with familial ALS.
About 20% of all familial ALS cases are known to have mutations involving the
SOD1 gene. Enzyme activity was reduced (by about 50%) in the red cells and in
the brain and spinal cord (10)
of patients with these mutations. Given the presence of SOD1 in almost all cellular
systems, the selective degeneration of motor neurons is a remarkable phenomenon
as yet unexplained.
Defective
Handling of Oxidative Stress
Cu/Zn SOD is a cytosolic enzyme that is known
to dismutate superoxide () generated intracellularly during oxidation
of various compounds, such as hypoxanthine to xanthine via xanthine oxidase
(83). This results in the formation of H2O2, which is then rapidly converted to H2O and O2 via the action of catalase, an enzyme present at high levels in most
cells. Also, oxidation of glutathione via glutathione peroxidase is another
way by which H2O2 can be converted to H2O. Given the high intracellular levels of glutathione,
this tripeptide may play an important role in cellular mechanisms protective against oxidative stress.
Malfunction of the
cytosolic SOD is expected to impair the ability of the cell to eliminate produced during certain oxidation reactions. The nondetoxified superoxide
radical can oxidize a number of cellular systems, particularly lipids that are constituents of the many membrane systems
present in each cell (83). Peroxidation of
cell membrane lipids, in particular, is expected to alter the membrane properties.
Membrane fluidity may be affected along with membrane permeability to various compounds. Because the cytoplasmic
membrane is essential for the transport of many biologically important substances,
these processes are expected to be impaired.
On theoretical grounds (66), an increased
membrane permeability to substances with high intracellular concentration, such
as glutamate, is
expected to lead to leakage of these substances out
of the cell, resulting in an increased
workload for the various membrane systems responsible for transporting these
substances against a concentration gradient. This, in turn, is expected to enhance
the metabolic demands of the cells and perhaps lead to increased superoxide
formation. Peroxidation of membrane lipids may also occur in other membranous
structures of the cell such as the endoplasmic reticulum (83), the Golgi apparatus, and the outer mitochondrial and
nuclear membranes. In this regard, it may be relevant that fragmentation of
the Golgi apparatus has been shown to occur in ALS (43). Also, oxidation of enzymes may impair energy metabolism
or other metabolic processes (83).
Given the limited ability of to penetrate
membranes, this oxidative damage may involve primarily cytosolic enzymes.
In
light of the above considerations, it is of interest that Ghadge et al. (26 ) showed that
expression of mutant SOD1 in PC12 cells is associated with higher rates of superoxide
production under various conditions. Hall et al. (27) detected an increase in spinal cord lipid peroxidation in transgenic mice with
SOD1 mutations. In addition, Shaw et al. (82) detected increased protein carbonyl
levels in the spinal cord of patients with sporadic motor neuron disease, thus indicating increased oxidative damage in
these patients.
With respect to
the effects of increased oxidative stress on energy metabolism, Browne et al.
(10) recently reported that ALS patients with SOD1 mutations
show marked increases in the mitochondrial Complex I and II-II activities. Similar
findings were also reported in transgenic mice overexpressing human mutant SOD1
(26).
Whether or not
these abnormalities involving mitochondrial energy
metabolism reflect an adaptation to enhanced energy demand, as postulated above,
it remains to be further studied.
Other investigators have focused their attention on structural proteins.
Crow et al. (15) suggested that SOD1-catalyzed
nitration of neurofilament-L may play a role in the ALS pathogenesis. Neurofilament
pathology has been identified in familial ALS cases associated with mutations
of neurofilament proteins. Bruijn et al. (11)
recently suggested that SOD1 mutations cause aggregation of the mutated protein. This, along with coaggregation of unidentified essential components
of the cell and/or aberrant catalysis by misfolded SOD1 mutant enzyme could
account for the MND pathology (11).
Although the SOD1 defect is limited to a small
percentage of ALS patients, its recognition is of paramount importance for the
following reasons: (a) A motor neuron disease, similar in all respects to sporadic
ALS, can result from a generalized metabolic abnormality, thus indicating that
such defects are capable of damaging motor neurons selectively; (b) the consequences
of SOD1 abnormalities on cellular functions are precisely those implicated in
the pathogenesis of primary ALS, which have been associated with altered glutamate metabolism
(increased membrane permeability, impaired transport, and/or energy metabolism);
and (c) there are probably myriad abnormalities with pathophysiological consequences
similar to those induced by SOD1 malfunction, and this is consistent with the
proposed multifactorial origin of ALS.
THE
PROBLEM OF SELECTIVE MOTOR NEURON VULNERABILITY
Although the defect in glutamate metabolism in
primary ALS is generalized, motor neurons are the only targets of the degenerative
process. The same is also true for the familial ALS, which has been linked to
Cu/Zn SOD mutations because this enzyme is expressed in many tissues. As such,
there is no reason to believe that the above metabolic abnormalities are limited
to the motor pathways. If this is so, why are only motor neurons affected?
The Glycine-Potentiated
Excitotoxic Hypothesis
Based
on the pattern of neuronal connectivity and the characteristics of glutamate
receptors, Plaitakis (63)
suggested that the glycinergic co-innervation renders motor neurons
the selective targets of a glutamate-mediated neurodegenerative process. In
motor neurons, unlike other neuronal systems, inhibition is in large part mediated
through glycinergic interneurons. Glycine
released from such glycinergic nerve terminals is known to inhibit motor neurons
by binding to a Cl- channel-linked glycinergic
receptor, which is strychnine-sensitive. In addition, glycine has been shown
to potentiate glutamatergic transmission by acting on a strychnine-insensitive
allosteric site of the NMDA subtype of glutamate receptor (37). Glycine increases
the frequency of the NMDA receptor channel openings by shortening the desensitization
period that follows glutamate action on this receptor (37). Desensitization of glutamate receptors seems to be of importance, since
it may represent the main mechanism
by which the neurotransmitter action of glutamate is terminated. Because both
dorsal and ventral horn neurons express NMDA receptors in high density
(80), the differential sensitivity of spinal cord
neurons to ALS neurodegeneration cannot solely relate to the presence of NMDA
receptors on these cells.
Studies
on cultured chick motor neurons have shown that innervation by interneurons
potentiates glutamate transmission (49). A substantial proportion of such interneurons are glycinergic, projecting
to motor neurons with glycine levels known to be greater in the ventral than
in the dorsal horns. There are also indications that motor neurons receive a
dense glutamatergic innervation (39). Mitchell
et al. (39) found that Na+-dependent glutamate transport, expected to be associated with glutamatergic
synaptic elements, is more densely
expressed in the ventral than the dorsal horns of the cat. Also, motor neurons that degenerate
in ALS are surrounded by astrocytes expressing high levels of the EAAT2 (39).
Although the glycine
allosteric site at the NMDA receptor is thought to be fully saturated by normal
levels of nerve tissue glycine, Thomson et al. (86) obtained enhancement of excitatory postsynaptic potentials
in cerebral cortex slices by applying increased concentrations of glycine. Similarly,
Budai et al. (12) obtained enhancement
of NMDA-evoked neuronal activity by glycine in the rat spinal cord in vivo
and concluded that the glycine sites on NMDA receptors were not saturated.
Under conditions
of enhanced synaptic glutamate, postsynaptic transduction seems to be mediated
primarily by the NMDA receptor. As such, desensitization of this receptor may
be of particular importance for protecting postsynaptic neurons from neurotoxicity.
In the motor neurons, however, the presence of high glycine levels may lead
to prolonged openings of the NMDA-linked channels, with resultant excessive entrance of Ca2+ into these cells. This, in turn, is expected
to activate intracellular proteases and lipases, which can damage the cell by
inducing a variety of secondary changes. Also, prolonged depolarization of postsynaptic
neurons by glutamate analogues is shown to cause ATP depletion and accumulation
of intracellular
purine metabolites (48). Depletion of ATP is thought to impair the function
of ion-dependent ATPases and thus membrane fluxes. Studies on kainate toxicity
in cerebellar slices showed that, in addition to ATP depletion, an increased
leakage of glutamate and aspartate occurred into the medium (48). However, the glycine hypothesis does not readily explain the degeneration
of corticospinal neurons as well as the sparing of the oculomotor neurons that
occur in ALS. In this regard, the pattern of NMDAergic and glycinergic synapses
in the motor cortex in health and disease remains to be further studied.
The
Glutamate Transporter Hypothesis
An alternative hypothesis suggested
by Medina et al (39) and Milton
et al (40) is that neuronal vulnerability in ALS relates to the level
of expression of glutamate transporters. These investigators (39,40)
found that motor neurons vulnerable to ALS neurodegeneration are surrounded
by astrocytes, which show a higher level of expression of EAAT2 than glial cells
present around motor neurons resistant to this neurodegeneration (oculomotor
nuclei). However, this hypothesis cannot explain the overall pattern of CNS
degeneration in ALS, since the highest levels of EAAT2 expression occurs in
the caudate nucleus, the nucleus basalis of Meynert and hippocampus (32).
These brain regions are not ordinarily involved in ALS.
Glutamate or Oxidative
Stress: Which Is Primary?
As
already discussed, altered glutamate metabolism has been shown in patients with
primary ALS, which accounts for the overwhelming majority
of patients with MND. These patients do not seem to have mutations of the SOD1
gene, which are specifically associated with familial ALS. Since the glutamatergic abnormalities detected in primary ALS suggest a defect in membrane permeability and since SOD1 malfunction could
damage membranes via lipid peroxidation (see above), it is possible that membrane abnormalities induced
by diverse etiologies may cause altered presynaptic glutamatergic mechanisms
and selective degeneration of motor neurons.
As indicated above, glycine-induced potentiation of NMDA function, could increase the metabolic demands of motor
neurons and/or make them bear, perhaps temporarily, increased Ca2+ loads. This may
lead to an increased production of superoxide radicals (38). ATP breakdown is thought to generate increased amounts
of hypoxanthine (38,
48), oxidation of which may lead to superoxide formation
(83). Normal cells may compensate by enhancing their
defenses against oxidative stress. However, motor neurons with SOD1 malfunction
may be less well able to handle oxidative stress. Over the years, accumulation
of superoxide radicals may damage cell membranes through lipid peroxidation,
causing altered glutamate distribution and thus initiating a vicious cycle.
Whether this explains the rapid downhill course patients often experience after
remaining disease-free for decades remains to be established.
There
is evidence that neuroexcitotoxicity is associated with increased oxidative
stress that may contribute to nerve cell death (44). Kainate-induced degeneration of cultured cerebellar
neurons was shown to be prevented by inhibiting xanthine oxidase or the formation
of this enzyme from xanthine dehydrogenase (38). Another mechanism by which glutamate toxicity may
lead to increased oxidative stress is inhibition of cystine transport with a
resultant reduction in intracellular glutathione levels (44). Conversely, failure of antioxidant mechanisms seems
to potentiate neuroexcitotoxic cell damage. Also, the pattern of motor neuron degeneration induced
by excitotoxin agonists is similar to that seen in the SOD-1 transgenic mouse
model of ALS (28). As such,
multiple metabolic abnormalities capable of interfering with different aspects
of glutamate transmission and/or cellular defenses against oxidative stress
may underlie primary ALS. The primary abnormality(ies) remains to be defined,
and therefore the questions raised by these observations are an important challenge
to modern neuroscience.
This work was supported by NIH grant NH-16871
and by Mount Sinai Research Center grant RR00071.
published 2000