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The Journal of Neuroscience, December 15, 1998, 18(24):10287-10296
Thiol Oxidation and Loss of Mitochondrial Complex I Precede
Excitatory Amino Acid-Mediated Neurodegeneration
Krishnan
Sriram1,
Susarla K.
Shankar2,
Michael R.
Boyd3, and
Vijayalakshmi
Ravindranath1
Departments of 1 Neurochemistry and
2 Neuropathology, National Institute of Mental Health and
Neurosciences, Bangalore 560 029, India, and 3 Laboratory
of Drug Discovery Research and Development, Developmental Therapeutics
Program, National Cancer Institute, Frederick Cancer Research and
Development Center, Frederick, Maryland 21702-1201
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ABSTRACT |
Human ingestion of "chickling peas" from the plant
Lathyrus sativus, which contains an excitatory amino
acid, L-BOAA
(L- -N-oxalylamino-L-alanine), leads to a progressive corticospinal neurodegenerative disorder, neurolathyrism. Exposure to L-BOAA, but not its optical
enantiomer D-BOAA, causes mitochondrial dysfunction as
evidenced by loss of complex I activity in vitro in male
mouse brain slices and in vivo in selected regions of
mouse CNS (lumbosacral cord and motor cortex). Loss of complex I
activity in lumbosacral cord after L-BOAA administration to
mice was accompanied by concurrent loss of glutathione. The inhibited
complex I activity in mitochondria isolated from lumbosacral cord of
animals treated with L-BOAA rebounded after incubation with
the thiol-reducing agent dithiothreitol, indicating that oxidation of
protein thiols to disulfides was responsible for enzyme inhibition. The
inhibition of complex I could be abolished by pretreatment with
antioxidant thiols such as glutathione ester and  -lipoic acid.
Chronic treatment of male mice, but not female mice, with
L-BOAA resulted in loss of complex I activity and
vacuolation and dendritic swelling of neurons in the motor cortex and
lumbar cord, paralleling the regionality of the aforementioned
biochemical effects on CNS mitochondria. These results support the view
that thiol oxidation and concomitant mitochondrial dysfunction (also
implicated in other neurodegenerative disorders), occurring downstream
of glutamate receptor activation by L-BOAA, are primary
events leading to neurodegeneration. Maintenance of protein thiol
homeostasis by thiol delivery agents could potentially offer protection
against excitotoxic insults such as those seen with
L-BOAA.
Key words:
excitatory amino acids; mitochondrial electron transport; NADH ubiquinone-1 oxidoreductase (complex I); brain; L-BOAA; oxidative stress; glutathione; protein thiol
oxidation
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INTRODUCTION |
L--Oxalylaminoalanine
(L-BOAA) is a naturally occurring nonprotein amino acid,
first isolated from chickling pea obtained from the plant
Lathyrus sativus (Rao et al., 1964 ) grown in the drought-prone areas of Africa and Asia. In humans, ingestion of the
chickling pea as a staple diet during famine results in a slowly
progressive, neurodegenerative condition known as neurolathyrism (Selye, 1957), a form of motor neuron disease. L-BOAA is
generally considered to be the causative agent in neurolathyrism
(Spencer et al., 1986). Neurolathyrism in humans is characterized by
spastic paraparesis that predominantly targets the Betz cells and the corticospinal tracts (Ludolph and Spencer, 1996). It is a highly prevalent neurotoxic disorder (Haimanot et al., 1990) that
predominantly affects males (Roy, 1988). Clinical (Cohn and Streifler,
1981) and neuropathological studies (Streifler et al., 1977) have shown the involvement of upper motor neurons, degeneration of anterior horn
cells, and loss of axons in the pyramidal tracts in the lumbar spinal
cord in humans affected by neurolathyrism.
L-BOAA exposure was first shown to cause seizures in
newborn mice in vivo and degeneration in CNS explant
cultures in vitro (Ross and Spencer, 1987), effects that
were abrogated in dose-dependent manner by known antagonists of a
subclass of glutamate receptor, namely, quisqualic receptors. The
unique action of L-BOAA on AMPA receptors was subsequently
identified (Ross et al., 1989), and the ability of the specific AMPA
receptor antagonist NBQX
(2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline) to prevent
L-BOAA-induced neuronal damage confirmed that
L-BOAA exerted its action through the AMPA receptors
(Pearson and Nunn, 1991). In synaptic membrane preparations,
L-BOAA prevented the binding of
[3H]AMPA, and the ability of L-BOAA to
displace [3H]AMPA from membranes prepared from
cerebral cortex exceeded that of quisqualic acid or AMPA itself
(Bridges et al., 1988; Ross et al., 1989). Although it is generally
accepted that L-BOAA causes neurodegeneration through
excitotoxic mechanisms, probably involving the AMPA receptors, the
actual molecular mechanisms involved in L-BOAA-induced
neurotoxicity generally remain uncharacterized.
Mitochondrial dysfunction has been implicated in a variety
neurodegenerative disorders. Abnormalities in complex I have been identified in mitochondria from platelet, brain, and muscle of Parkinson's disease patients (Parker et al., 1989; Mizuno et al., 1995), and cytochrome c oxidase deficiency has been observed in Alzheimer's disease brain (Parker et al., 1994). The present studies indicate that an inhibition of mitochondrial complex I activity mediated through oxidation of thiol groups in distinctive regions of
the CNS is a primary event in thiol-reversible,
L-BOAA-induced toxicity.
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MATERIALS AND METHODS |
Materials. L-BOAA, L-BMAA,
AMPA, kainic acid, quisqualic acid, and MK-801 were obtained from
Research Biochemicals (Natick, MA). NMDA, N-acetyl aspartyl
glutamate (NAAG), and glutamic acid were obtained from Sigma (St.
Louis, MO). D-BOAA was procured from Tocris Neuramin and as
a gift from Dr. S. L. N. Rao, Osmania University, Hyderabad,
India. NBQX and ubiquinone-1 were gifts from Novo Nordisk and Eisai
Pharmaceutical Company, respectively. Glutathione isopropyl ester (GSH
ester) was obtained as a gift from Yamanouchi Pharmaceutical Company.
All other chemicals and reagents were of analytical grade and were
obtained from Sigma or Qualigens.
Animals. Male and female Swiss albino mice (3-4
months old; 25-30 gm) or male Wistar rats (3-4 months old, 200-250
gm) were obtained from National Institute of Mental Health and
Neurosciences (NIMHANS) Animal Research Facility. Animals had access to
pelleted diet (Lipton, Calcutta, India) and water ad
libitum.
In vitro studies. Sagittal slices of male mouse or rat
brain were prepared using the previously described apparatus
developed in our NIMHANS laboratory (Pai et al., 1991). Animals were
decapitated, and the brains were quickly removed and kept on a flat
surface at 4°C. The slicer was placed above the brain with equal
number of blades on either side of the median plane and gently pressed. The slices were carefully transferred using a flat-edged needle to a
beaker containing artificial CSF (ACSF). Four slices of uniform thickness (1 mm) were prepared from each mouse brain in a time not
exceeding 1-2 min. Slices were incubated in ACSF, pH 7.4, containing
sodium chloride (122 mM), potassium chloride (3.1 mM), calcium chloride (1.3 mM), magnesium
sulfate (1.2 mM), glucose (10 mM), and glycyl
glycine (30 mM) in an oxygen-enriched atmosphere as given
by McIlwain (1975) with or without L-BOAA for 1 hr at 37°C.
In experiments performed to evaluate the effects of antioxidant thiols
such as glutathione isopropyl ester (1 mM) or
-lipoic acid (100 µM); slices were preincubated
with the agents for 30 min at 37°C, before exposure to
L-BOAA (1 nM) for 1 hr. To assess the
protective effects of glutamate receptor antagonists on
L-BOAA-mediated neurotoxicity, sagittal slices of male
mouse brain were prepared from animals dosed 4 hr earlier with NBQX (30 mg/kg body weight, i.p.) and incubated without or with
L-BOAA (1 nM).
In vivo studies. L-BOAA was dissolved in
normal saline (5 or 10 mg/kg body weight, s.c.) and administered to
male and female Swiss albino mice. Control animals received vehicle
alone. The animals were killed at specified time points, and the
brain and spinal cord were removed. Kidney, heart, lung, and the main
lobe of the liver were also removed for certain experiments. All
dissections and tissue handling were performed at 4°C. The motor
cortex (Fig. 1) was dissected out from
the brain. The spinal cord was exposed, and the thoracic cord was
collected. The lumbar and sacral segments of the cord were dissected
out together and used for experiments. In some experiments, the cortex
was dissected into three segments, namely, the frontal and parietal
cortex, temporal cortex, and occipital cortex, following anatomical
landmarks. In experiments performed to assess the effect of the AMPA
receptor antagonist NBQX, mice were treated with NBQX (30 mg/kg body
weight, i.p., in saline). After 4 hr, L-BOAA (10 mg/kg body
weight, s.c., in saline) was administered. Control animals received
vehicle alone. In some experiments, the thiol antioxidant -lipoic
acid (20 mg/kg body weight, s.c) was given 0.5 hr before
L-BOAA. All animals were killed 24 hr after
L-BOAA injection, and the lumbosacral cord was
dissected.
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Figure 1.
The region from the mouse brain cortical layer
(shaded area) that was dissected out as motor cortex for
measurement of enzyme activity in all subsequent experiments
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L-BOAA was also administered chronically (5 mg/kg body
weight, s.c., daily) to male and female mice, and animals were killed after 5, 10, 20, or 40 d. After 40 d, L-BOAA
administration was discontinued and the animals were killed on the 55th
day after the first dose of L-BOAA. The motor cortex,
thoracic, and lumbosacral segments of the spinal cord were collected
for assay of complex I.
Processing of tissue. The tissues (brain slices and cortical
regions of mouse brain or spinal cord segments or other tissue) were
processed immediately for the assay of activities of complex I, complex
II, complex II-III, complex IV, and concentration of glutathione. For
the assay of mitochondrial electron transport complexes, tissues were
homogenized in sucrose (0.25 M) and centrifuged at
1000 × g for 10 min to obtain a postnuclear
supernatant, which was recentrifuged at 17,000 × g for
20 min to obtain a crude mitochondrial pellet. The pellet was
resuspended in 0.25 M sucrose and freeze-thawed three times
before assay of complex I (Shults et al., 1995), complex II (Hatefi and
Stiggall, 1976), complex II-III (King, 1967), or complex IV (Gibson and
Hill, 1983).
Assay of NADH: ubiquinone oxidoreductase (complex I)
activity. Complex I activity was assayed in brain mitochondrial
preparations as rotenone-sensitive NADH:ubiquinone-1 oxidoreductase
according to Shults et al. (1995) with minor modifications as described below. The assay was performed in phosphate buffer (35 mM,
pH 7.2) containing sodium cyanide (2.65 mM), magnesium
chloride (5 mM), EDTA (1 mM), bovine serum
albumin (1 mg/ml), and antimycin (2 µg/ml). Brain mitochondria
(90-150 µg of protein), coenzyme Q-1 (ubiquinone-1, 0.05 mM final concentration) were added to the assay buffer such
that the final assay volume was 0.48 ml. After preincubation of the
reaction mixture at room temperature for 2 min, the reaction was
initiated by the addition of 0.02 ml of a 5 mM solution of
NADH. The decrease in absorbance at 340 nm was monitored over time. The
assay was also performed in the presence of rotenone (5 µM final concentration) to determine the rotenone-insensitive complex I activity. The rotenone-sensitive enzyme
activity was calculated by subtracting the rotenone-insensitive activity from the total activity and is expressed as nanomoles of NADH
oxidized per minute per milligram of protein.
Estimation of glutathione. For estimation of glutathione,
the removed tissues were frozen immediately in liquid nitrogen and homogenized in potassium phosphate buffer (100 mM, pH 7.4)
containing 1 mM EDTA. Total glutathione levels were
estimated according to the method described by Tietze (1969) as follows
briefly. To an aliquot of the brain homogenate, an equal volume of
5-sulfosalicylic acid (1%, w/v) was added, mixed, and centrifuged at
10,000 × g for 10 min, and the supernatant was
collected and used for assay of GSH by the enzymatic recycling method.
Another aliquot of the brain homogenate was used for protein
estimation. Protein was estimated by a dye-binding method (Bradford,
1976). Statistical analyses were performed using Student's
t test or ANOVA followed by Student-Newman-Keuls,
Bonferroni, or Dunnet's test.
Histology. Brain and total length of the spinal cord from
control and mice treated with L-BOAA were fixed by
perfusion with Bouin's fixative, and representative sections were
paraffin-embedded, sectioned, and stained with hemotoxylin and eosin or
cresyl violet for Nissl.
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RESULTS |
In vitro studies
Exposure of sagittal slices of male mouse brain to varying
concentrations of L-BOAA resulted in a dose-dependent
inhibition of complex I. Incubation of slices with 0.1 pM
L-BOAA inhibited complex I by 28%; at the highest
concentration of L-BOAA tested (1 nM), the
enzyme activity was inhibited by ~50% (Fig.
2A). Complex I was
significantly inhibited (17.8% inhibition) within 5 min of exposure to
the toxin (Fig. 2B), and, after 60 min of incubation with L-BOAA, the enzyme activity was inhibited by 51%
(Fig. 3). D-BOAA at a similar
or even higher concentration had no effect on complex I activity,
indicating the stereospecificity of the L-isomer in
producing toxicity (data not shown).
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Figure 2.
Effect of L-BOAA on complex
I activity in mouse and rat brain slices. A, Sagittal
slices of mouse or rat brain were incubated in ACSF with varying
concentrations (0.01 pM to 1 µM) of
L-BOAA for 1 hr, and the activity of complex I was assayed
in the crude mitochondrial preparation. B,
Time-dependent inhibition of complex I activity by L-BOAA
in mouse and rat brain slices. Sagittal slices of mouse brain were
incubated in ACSF without ( -) or with L-BOAA (1 nM,  -) for varying intervals (5-60 min), and the
activity of complex I was assayed in the crude mitochondrial
preparation. The enzyme activity is expressed as nanomoles of
NADH oxidized per minute per milligram of protein. Values are mean ± SEM (n = 4-6 slices).
Asterisks indicate values significantly different from
respective control (p < 0.01).
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Figure 3.
Effect of L-BOAA on GSH levels in
mouse brain slices (A) and effect of GSH on
complex I activity inhibited by L-BOAA
(B). A, Sagittal slices of mouse
brain were incubated in ACSF without (-) and with
L-BOAA (1 nM,  -) for varying intervals
(5-30 min). GSH levels are expressed as nanomoles of GSH per milligram
of protein. Values are mean ± SEM (n = 13-17
slices). B, Mitochondria were prepared from untreated
and L-BOAA-treated mouse brain slices, and complex I
activity was assayed. Two aliquots of this preparation were incubated
with and without GSH (2 mM) for 30 min at 37°C.
Thereafter, the activity of complex I was assayed and expressed as
nanomoles of NADH oxidized per minute per milligram of protein. Values
are mean ± SEM (n = 5, wherein each sample
was prepared by pooling mitochondria from 3 slices).
Asterisks in A and B
denote values significantly different from respective controls
(p < 0.05).
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A marked difference in the response to toxicity was observed when brain
slices prepared from male rats and mice were exposed to
L-BOAA. The minimum toxic dose required to inhibit complex I significantly in rat brain slices was 1 nM (Fig.
2A), as compared with the minimum toxic dose of 0.01 pM required to inhibit the enzyme in mouse brain slices.
To evaluate if L-BOAA had any effect on other mitochondrial
respiratory chain enzymes, thereby contributing to mitochondrial dysfunction, we assessed the activities of some of these enzymes in
male mouse brain slices incubated with L-BOAA for 1 hr.
L-BOAA (1 nM) significantly inhibited the
activities of succinate dehydrogenase (complex II) and
succinate-cytochrome c oxidase (complex II-III) by 28 and 30%,
respectively. The concentration of L-BOAA at which these
enzymes were inhibited was profoundly higher (1 nM) than that required to inhibit complex I (0.01 pM).
L-BOAA had no effect on cytochrome c oxidase (complex IV)
activity even at the highest tested concentration (data not shown).
The neurotoxic effect of L-BOAA (0.1 pM) was
compared with other glutamate agonists: L-glutamic acid (10 µM and 1 mM), NAAG (0.1 pM and 1 µM), NMDA (1 nM and 1 µM),
quisqualic acid (1 nM and 1 µM), AMPA (100 µM and 1 mM), kainic acid (1 µM
and 1 mM), or L-BMAA (1 mM). The lower
concentrations of the agonists selected represent the minimum toxic
dose that was required to induce significant leakage of lactate
dehydrogenase from the slices into the medium (Pai and Ravindranath,
1993). In addition, higher concentrations (10- to 100-fold) of
these agonists were also tested to determine their effect on
mitochondrial function. Only glutamic acid and AMPA significantly
inhibited complex I, albeit at millimolar concentrations; the other
agonists tested had no effect on complex I activity at any of the
concentrations tested (Table 1).
Exposure of male mouse brain slices to L-BOAA (1 nM) resulted in a small, transient decrease (8.7%) of the
endogenous thiol antioxidant GSH 10 min after incubation with the toxin
(Fig. 3A). The GSH levels recovered to control values within
15 min and were not significantly different from controls at all other
time points. To determine whether the loss in complex I activity was
caused by oxidation of protein thiols, mitochondria prepared from
L-BOAA-treated slices were incubated without and with GSH
(2 mM). In mitochondrial incubations containing GSH,
complex I activity was restored, whereas in incubations with buffer
alone, the enzyme activity remained inhibited (Fig. 3B),
suggesting the involvement of thiol oxidation as a cause of complex I
inhibition by L-BOAA.
To determine the mechanism of complex I inhibition by
L-BOAA, experiments were designed to assess if previous
exposure to thiol antioxidants could protect against L-BOAA
neurotoxicity. Pretreatment of male mouse brain slices with GSH
isopropyl ester (1 mM) 0.5 hr before exposure to the toxin
(1 nM) protected against L-BOAA-mediated
inhibition of complex I (Table 2).
Similarly, pretreatment with -lipoic acid (100 µM)
also afforded protection, indicating that inhibition of complex I may
be caused by the loss of thiol homeostasis (Table 2). The AMPA receptor
antagonist NBQX also afforded protection against
L-BOAA-mediated inhibition of complex I in mouse brain
slices (Table 2).
In vivo studies
In male mice given a single dose of L-BOAA (10 mg/kg
body weight, s.c.), selective loss of mitochondrial complex I activity was observed in the frontoparietal cortex (Fig.
4A, wherein the motor
cortex is located, Fig. 1) and the lumbosacral segment of the spinal
cord (Fig. 4B) within 0.5 hr after administration. Complex I activity was not significantly decreased (Fig.
4A-D) in other brain regions (temporal
and occipital cortex, striatum, hippocampus, brainstem, thalamus, and
cerebellum), other segments of spinal cord (cervical and thoracic cord)
and in non-CNS tissue (heart, lung, kidney, and liver). In all further
experiments, the area depicted in Figure 1 was designated as motor
cortex and was dissected out for biochemical estimations.
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Figure 4.
Effect of L-BOAA administration on
complex I activity in different regions of mouse brain cortex
(A), different segments of spinal cord
(B), various regions of brain
(C), and in other organs
(D) of mice. Mice were administered a single dose
of L-BOAA (10 mg/kg body weight, s.c.) and killed 0.5 hr
after the injection ( ). Controls received vehicle alone (saline,
). Mitochondria prepared from the tissues were used to measure
complex I activity. F/P CT,
Frontoparietal cortex; T CT, temporal cortex; O
CT, occipital cortex; C CD, cervical cord;
T CD, thoracic cord; L/S
CD, lumbosacral cord; ST, striatum;
HP, hippocampus; BS, brainstem;
TH, thalamus; CE, cerebellum;
HT, heart; LG, lung; KD,
kidney; LV, liver. Values are mean ± SEM
(n = 4 animals). Asterisks indicate
values significantly different from respective controls
(p < 0.01).
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Administration of the optical antipode of the glutamate agonist,
namely, D-BOAA (5 and 10 mg/kg body weight, s.c.) to male mice resulted in no change in complex I activity in either lumbosacral region of the spinal cord or motor cortex (Fig.
5A,B,
respectively). In contrast, administration of the same dose of
L-BOAA resulted in significant inhibition of complex I
activity in both regions of the CNS within 0.5 hr. Although the enzyme
activity in the motor cortex was inhibited (Fig. 5B) after
administration of the lower dose of L-BOAA (5 mg/kg body
weight), the activity in the lumbosacral cord was affected only after
administration of higher dose of the glutamate agonist (Fig.
5A).
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Figure 5.
Effect of administration of varying
doses of D-BOAA and L-BOAA on complex I
activity in mouse brain lumbosacral segment of spinal cord
(A) and motor cortex (B).
Mice were administered varying doses of D-BOAA or
L-BOAA (5 or 10 mg/kg body weight, s.c). Control animals
received vehicle alone. The animals were killed 0.5 hr after
administration of amino acids. The motor cortex
(B) and lumbosacral cord
(A) were dissected out and complex I
activity was measured in the mitochondrial preparation. Values are
mean ± SEM (n = 3-5 animals).
Asterisks indicate values significantly different
from control (p < 0.05).
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When we followed the effect of L-BOAA on complex I activity
in motor cortex and lumbosacral cord over a time period, a striking difference was noticed. A sustained loss of enzyme activity (Fig. 6B) was observed in the
lumbosacral cord after a single dose of L-BOAA (10 mg/kg
body weight), whereas in the motor cortex, the enzyme activity was
inhibited at 0.5 hr after the dose, but rebounded at 1 hr and returned
to near normal levels by 4 hr. However, at the end of 24 hr, complex I
activity remained inhibited (Fig. 6A). Under the
same conditions, the enzyme activity in the thoracic segment of the
cord was unaffected. Complex I activity was also unaffected in the
hippocampus and striatum after L-BOAA administration (5 or
10 mg/kg body weight).
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Figure 6.
Complex I activity and GSH concentration
in the motor cortex (A) and lumbosacral cord
(B) at various time periods after a single dose
of L-BOAA. Mice were administered L-BOAA (10 mg/kg body weight, s.c.), whereas control animals received vehicle
alone. The animals were killed at indicated time periods. The motor
cortex (A) and the lumbosacral segment of the
spinal cord (B) were dissected out and used for
the assay of complex I (in mitochondrial preparation) or GSH (in tissue
homogenate). The control values were similar at all time points
examined. Values are mean ± SEM (n = 4 animals). Asterisks indicate values significantly
different from corresponding control (p < 0.05).
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Complex II activity in both motor cortex and spinal cord was inhibited
only 4 and 1 hr after the L-BOAA dose, respectively, while
remaining unaffected in the thoracic cord. Thus, inhibition of complex
II was observed several hours after the inhibition of complex I (data
not shown).
We measured the total GSH content and complex I activity in motor
cortex (Fig. 6A) and lumbosacral cord (Fig.
6B) over a 24 hr period after a single dose of
L-BOAA (10 mg/kg body weight). A small but significant
decrease in GSH levels was seen in the motor cortex at 0.5 and 1 hr
after the administration of L-BOAA. The GSH levels,
thereafter, were similar to control levels. Interestingly, in the motor
cortex, the complex I activity showed a transient decrease 0.5 hr after
L-BOAA dose, and thereafter rebounded to enzyme activity
higher than controls, however, after 24 hr, complex I activity in the
motor cortex was significantly lower than controls, indicating that a
single dose of L-BOAA resulted in a long-term inhibition
(Fig. 6A). In the lumbosacral region of the spinal cord, sustained loss of both complex I and GSH was observed from 0.5 to
24 hr after the administration of a single dose of L-BOAA (Fig. 6B). It was only 48 hr after administration of
a single dose of L-BOAA that both GSH and complex I
returned to near control levels in both motor cortex and lumbosacral
cord (data not shown). Mitochondria from the motor cortex (Fig.
7A) and lumbosacral region of
spinal cord (Fig. 7B) of mice treated with
L-BOAA (10 mg/kg body weight) were incubated in
vitro with the thiol reductant dithiothreitol, and complex I
activity was assayed. Dithiothreitol completely reversed the action of
L-BOAA and restored the complex I activity to levels
comparable with those in corresponding controls (Fig.
7A,B), indicating that the
inhibition of enzyme activity was caused by oxidation of thiols to
protein disulfides.
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Figure 7.
Reversal by dithiothreitol of
L-BOAA-mediated inhibition of complex I in the motor cortex
(A) and lumbosacral cord
(B). Mice were administered L-BOAA
(10 mg/kg body weight, s.c.) and killed 0.5 hr after dosing.
Mitochondria, prepared by pooling the tissue from three animals, were
freeze-thawed three times, and complex I activity was assayed.
Thereafter, mitochondria from each sample were divided into two
aliquots and incubated with and without dithiothreitol (3.5 mM) for 30 min at 37°C. After the incubation, the samples
were dialyzed, and the activity of complex I was assayed before and
after incubation with dithiothreitol. Values are mean ± SEM
(n = 5 samples wherein the mitochondria prepared
from three animals were pooled to make one sample).
Asterisks indicate values significantly different from
control (p < 0.05).
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Pretreatment of mice with the AMPA receptor antagonist NBQX (30 mg/kg
body weight, i.p.) 4 hr before L-BOAA administration completely abolished L-BOAA-induced (10 mg/kg body weight,
s.c.) complex I inhibition in the lumbosacral cord, indicating that the
mitochondrial enzyme loss was mediated through downstream events of
glutamate receptor activation (Fig. 8,
top). Interestingly, pretreatment of mice with the thiol
antioxidant -lipoic acid also abolished L-BOAA-mediated
inhibition of complex I activity (Fig. 8, bottom),
indicating that thiol antioxidant could potentially reverse the
neurotoxic effects of L-BOAA.
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Figure 8.
Effect of pretreatment of mice with NBQX
(top) and -lipoic acid (bottom) on
complex I activity in lumbosacral cord after L-BOAA
administration. Top, Two sets of mice were administered
NBQX (30 mg/kg body weight, i.p.) and, after 4 hr, one set was dosed
with L-BOAA (10 mg/kg body weight, s.c.), whereas the other
set received vehicle (saline) alone. Bottom, Two sets of
mice were pretreated with -lipoic acid (20 mg/kg body weight, s.c.)
and, after 1 hr, one set was given L-BOAA as described in
the top panel. Another group of mice received only
L-BOAA, and the control animals received vehicle alone. The
animals were killed 24 hr after L-BOAA administration, the
lumbosacral segment of spinal cord was dissected out, and complex I
activity was estimated in the isolated mitochondria. Values are
mean ± SD (n = 4 animals).
Asterisks denote values significantly different from
control (p < 0.01).
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Neurolathyrism is known to occur in humans after consumption of at
least 300 gm/d of the chickling peas from the plant Lathyrus sativus for a period of at least 3 months (Streifler et al.,
1977). The concentration of L-BOAA in the pea varies from
0.1 to 2.5% (Roy, 1988) Assuming the average body weight of humans as
60 kg and the least L-BOAA concentration in the pea as
0.1% (w/w), the daily intake of L-BOAA would be 5 mg/kg
body weight. Male mice were administered 5 mg/kg body weight of
L-BOAA chronically for up to 40 d, after which one
group of animals was maintained untreated for an additional 15 d
before killing. Complex I activity in the lumbosacral cord (Fig.
9) showed marked reduction after 5, 10, 20, and 40 d of L-BOAA exposure (19, 29, 20, and 29%
decrease), whereas in the motor cortex (Fig. 9), the activity was
significantly decreased (48%) only after 40 d. After withdrawal
of L-BOAA, the complex I activity in the lumbosacral cord
recovered to control levels while remaining significantly depressed in
the motor cortex (Fig. 9).
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Figure 9.
Effect of chronic administration of
L-BOAA on complex I activity in thoracic (top
panel), lumbosacral segment of mouse spinal cord (middle
panel) and motor cortex (top
panel). Mice were administered L-BOAA
(5 mg/kg body weight. s.c., daily) and killed on days 5, 10, 20, and 40 (-). L-BOAA administration was stopped after 40 d, and the animals were kept on normal diet for 15 d before
killing. Control animals received vehicle alone (-). After
killing, the motor cortex (top), thoracic, and
lumbosacral segments of the spinal cord (middle) were
removed and assayed for complex I activity. Values are mean ± SD
(n = 6 animals). Asterisks denote
values significantly different from control
(p < 0.01).
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Morphological evaluation was performed on the brain and spinal cord of
animals that received L-BOAA for 40 d (Fig.
10A-D). Pyramidal neurons of layers 3 and 5 in the motor cortex revealed cytoplasmic vacuolation and swelling of the apical dendrites. Similar
pathology was also noted in the neurons of the motor cranial nerve
nuclei. In the spinal cord, the brunt of the pathology was seen in the
lumbar and sacral segments, with relative sparing of the cervical and
thoracic segments. The medial and intermediate group of neurons
corresponding to laminae 7 and 8 of the gray horn in the lumbar level
of the spinal cord were abnormal, whereas the lateral group of neurons
(lamina 9 of the gray horn) were relatively preserved. The spinal
neurons showed cytoplasmic vacuolation, dendritic swelling, and
depletion of Nissl substance. The neuronal contour was distorted
significantly in some by multiple vacuolation. However, the nucleus and
nucleolus were intact. Some of the vacuoles had a central, dark
granule. An increase in astrocytes was noted, many of them having a
pale watery nucleus and multiple obvious nucleoli, unlike in controls.
These astrocytes were seen as small clusters in the neuropil. In mice,
wherein L-BOAA was administered for 40 d and then
discontinued for 15 d, the pathological changes of neuronal
vacuolation receded.
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Figure 10.
Lumbar cord (A,
B) and motor cortex (C, D)
sections from control animals and those treated with L-BOAA
(5 mg/kg body weight for 40 d). A, Anterior horn
neurons of the lumbar spinal cord from control animal.
B, Similar section as A from animals
chronically administered L-BOAA; anterior horn neurons show
cytoplasmic vacuolation (arrowheads), whereas the
nucleus and nucleolus are relatively well preserved. The vacuolation of
the apical dendrites resulting in distortion of the neuronal contour is
seen at higher magnification in inset I; inset
II depicts the pale area in cytoplasm of the large neuron that
extends along the dendrite and indents the adjacent cell.
C, Cortical pyramidal neurons in layer V of motor cortex
from control mouse. D, A similar section as
C from L-BOAA-treated mouse showing
vacuolation and clearing along the apical dendrites
(arrowheads) and depletion of Nissl substance in the
pyramidal neurons. Scale bar, 25 µm.
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The effect of L-BOAA on mitochondrial complex I was
gender-specific. Exposure of brain slices from female mice to
L-BOAA had no effect on the complex I activity even at
concentrations up to 1 µM (data not shown).
Administration of a single dose of L-BOAA (10 mg/kg body
weight, s.c.; Fig.
11A) or chronic
exposure to L-BOAA (5 mg/day, s.c; Fig.
11B) for 40 d did not have any effect on the complex I activity in the motor cortex or lumbosacral cord, whereas the
activity was significantly inhibited in similarly treated groups of
male mice.
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Figure 11.
Effect of L-BOAA on mitochondrial
complex I in male and female mice. A, Male and female
mice were administered a single dose of L-BOAA (10 mg/kg
body weight) and killed 24 hr later. B, Male and female
mice were treated daily with L-BOAA (5 mg/kg body weight,
s.c.) for 40 d. Complex I activity was estimated in the motor
cortex (MC) and lumbosacral cord (LSC) in
vehicle-treated () and L-BOAA-treated () animals.
Values are mean ± SEM (n = 5-6 animals).
Asterisks indicate values significantly different from
corresponding controls (p < 0.05).
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DISCUSSION |
Animal models for neurolathyrism that mimic the progressive
degeneration of Betz cells in the motor cortex and the corticospinal tract have not been reported. Monkeys fed 10- to 20-fold higher doses
than that necessary to produce irreversible neurolathyrism in humans
develop the initial signs of the disease as evidenced by central motor
deficits involving the corticospinal tracts (Ludolph and Spencer,
1996). Administration of large doses of the toxic principle
L-BOAA to rodents, however, results in seizures
(ShashiVardan et al., 1997). Pathological effects in the animal model
of L-BOAA toxicity defined in the present study share
remarkable resemblance to the regiospecific effects seen in the human
disease. Selective inhibition of mitochondrial complex I (Fig. 4) and
pathological changes (Fig. 10) were seen in the lumbosacral cord and
the motor cortex after acute or chronic exposure to L-BOAA
with no apparent changes in all the other regions of the CNS that were
examined. The similarity with the human disease was also seen in the
gender-specific effects of L-BOAA; the female mice were
relatively resistant to the toxic effects (Fig. 11). In humans, the
incidence of neurolathyrism is more common in men, whereas women are
less prone to the disease, although the intake of the chickling pea
from Lathyrus sativus is not significantly different (Roy,
1988). It has been reported that pretreatment of cortical neuronal
cultures with estrogen protects them from excitotoxic insults when the
cells were challenged with glutamate (Behl et al., 1997). Although the
mechanism by which estrogens protect the neuronal cell is not known, it
is hypothesized that estrogen-like compounds, in general, are able to
protect the neurons from damage by oxidative stress-mediated events.
The inhibition of complex I is a sensitive and selective index of
L-BOAA toxicity. In vitro, in brain slices,
complex I is inhibited by very low concentrations of L-BOAA
in a dose- and time-dependent manner, an effect not shared by other
glutamate agonists tested, with the exception of AMPA and glutamate,
which inhibit complex I at very much higher doses. We had earlier
reported that L-BOAA inhibits NADH-dehydrogenase activity
in vitro in mouse brain slices (Pai and Ravindranath, 1993),
however, measurement of rotenone-sensitive complex I activity is a more
sensitive and selective index of L-BOAA toxicity.
The mechanism of inactivation of complex I seems to be a downstream
event of L-BOAA action on excitatory amino acid receptors, because the loss in complex I activity was blocked both in
vitro and in vivo by specific antagonists of AMPA
receptor. The neurotoxic effects of L-BOAA seem to be
predominant in the motor cortex and lumbosacral region of the spinal
cord, and this specificity does not seem to reflect the distribution of
AMPA receptors in the CNS. The observations made herein do seem to
suggest that hitherto undetected high-affinity binding sites for
L-BOAA may exist in the CNS. Earlier studies have shown
that administration of 14C-L-BOAA to rats
resulted in maximal accumulation of the radiolabel in the spinal cord
(Rao, 1978). Nevertheless, the regions of the CNS affected by
L-BOAA bear a remarkable similarity to the regions affected
in human forms of the disease.
The results described herein provide the first evidence that
L-BOAA, an excitatory amino acid, can cause mitochondrial
dysfunction through inhibition of complex I in vivo in
specific regions of the CNS. Furthermore, the inhibition of complex I
activity was associated with oxidation of protein thiol groups in
complex I (Dupius et al., 1991), because the enzyme activity rebounded
after incubating the mitochondria with the thiol-reducing agent
dithiothreitol. From the experimental evidence presented in this study,
wherein the oxidative stress generated (measured as loss of cellular
antioxidant thiol GSH) followed the inhibition of complex I activity
(Fig. 6); loss of mitochondrial complex I seems to be a primary event after exposure to L-BOAA. But because selective cell
populations are affected within specific regions of the brain by
L-BOAA, the measurement of GSH in sagittal slices of whole
brain as performed in the in vitro experiments may not
reflect subtle changes in the antioxidant status in the targeted cells.
However, the reversal of the loss in complex I by thiol reductants
seems to indicate that oxidative stress and protein thiol oxidation may
be primary events in L-BOAA induced mitochondrial
dysfunction. One of the downstream events of excessive stimulation of
glutamate receptors is the generation of reactive oxygen species
leading to oxidative stress-mediated cellular damage (Murphy et al.,
1989).
Mitochondrial dysfunction has been recognized as a primary event in
glutamate neurotoxicity (Schinder et al., 1996), particularly that
mediated through NMDA receptors. The activation of NMDA receptors leads
to massive increase in intracellular calcium, which results in the
alteration in the mitochondrial membrane potential and opening of the
mitochondrial permeability transition pore (White and Reynolds, 1996).
However, pore-opening through voltage-dependent channels is directly
associated with an oxidation-reduction-sensitive dithiol, and the
cross-linking of the sulfhydryl groups increases the probability of the
mitochondrial permeability transition pore opening, an event that can
be blocked by the thiol reductant dithiothreitol (Petronilli et al.,
1994). Thus, the mitochondrial dysfunction caused through mitochondrial
permeability transition pore opening is also sensitive to the oxidation
of protein thiols in a manner similar to that seen in the present study
with reference to the inhibitory effect of L-BOAA on
complex I. We have earlier shown that brain mitochondria (Ravindranath
and Reed, 1990) are severalfold more vulnerable to oxidative
stress-mediated damage as compared with liver mitochondria (Olafsdottir
and Reed, 1988), and the major consequence of oxidative stress on brain
mitochondria is the formation of protein-glutathione-mixed disulfide
(rather than increase in levels of oxidized GSH, GSSG as seen in liver
mitochondria). Taken together these studies demonstrate the
vulnerability of brain mitochondrial proteins to oxidative modification
through formation of mixed disulfides.
The involvement of excitatory amino acids in the pathogenesis of motor
neuron disease has been demonstrated both in sporadic cases (Rothstein
et al., 1990) and in chemically induced forms of the disease (Spencer
et al., 1986, 1987). More recent developments include the discovery of
mitochondrial dysfunction in a variety of neurodegenerative disorders,
including motor neuron disease (Chang et al., 1995; Mizuno et al.,
1995). This study provides evidence indicating a mechanism for
neurodegeneration through mitochondrial dysfunction chemically induced
through excitatory amino acids. Increased glutaminergic activity has
been shown to be associated with Parkinson's disease (Verma and
Kulkarni, 1995), and glutamate antagonists (Stauch Slusher et al.,
1995) have been shown to alleviate Parkinsonism in experimental
animals. Recent hypotheses on role of excitatory amino acids in
pathogenesis of sporadic motor neuron disease have also resulted in new
putative therapies that include anti-excitotoxicity agents (Rothstein, 1996). Although the loss of complex I activity has been demonstrated in
Parkinson's disease patients (Parker et al., 1989); the present study
indicates a similar dysfunction in chemically induced motor neuron
disease, in experimental animals. It remains to be seen if similar
dysfunction occurs in humans in sporadic forms of the disease.
In conclusion, the present investigation demonstrated that excitatory
amino acids like L-BOAA cause mitochondrial dysfunction in
selected regions of the CNS that can be reversed in vitro by thiol reductants. The results indicate an initiating role for oxidative
stress and the potential importance of maintaining protein thiol
homeostasis in brain through administration of thiol antioxidants.
| |
FOOTNOTES |
Received May 26, 1998; revised Sept. 21, 1998; accepted Sept. 28, 1998.
This work was supported by a grant from the United States-India fund
for cultural, educational, and scientific cooperation. We thank Dr.
S. L. N. Rao, Osmania University, Hyderabad, India for the
generous gift of D-BOAA.
Correspondence should be addressed to Dr. V. Ravindranath, Department
of Neurochemistry, Hosur Road, National Institute of Mental Health and
Neurosciences, Bangalore 560 029, India.
| |
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Top
Abstract
Introduction
