Abbreviations used: ALS, amyotrophic lateral sclerosis; DOPAC, 3,4-dihydroxyphenylacetic acid; EAAT, excitatory amino acid transporter; ECF, extracellular fluid; EFL-glu, extraction fraction for glutamate corrected for mannitol; 5-HIAA, 5-hydroxyindole-3-acetic acid; SOD1, Cu/Zn superoxide dismutase.
Elevated Cortical Extracellular Fluid Glutamate in Transgenic Mice Expressing Human Mutant (G93A) Cu/Zn Superoxide Dismutase
Article first published online: 18 JAN 2002
Journal of Neurochemistry
Volume 74, Issue 4, pages 1666–1673, April 2000
How to Cite
Alexander, G. M., Deitch, J. S., Seeburger, J. L., Valle, L. D. and Heiman-Patterson, T. D. (2000), Elevated Cortical Extracellular Fluid Glutamate in Transgenic Mice Expressing Human Mutant (G93A) Cu/Zn Superoxide Dismutase. Journal of Neurochemistry, 74: 1666–1673. doi: 10.1046/j.1471-4159.2000.0741666.x
- Issue published online: 18 JAN 2002
- Article first published online: 18 JAN 2002
- Amyotrophic lateral sclerosis;
Abstract: Transgenic mice expressing a mutated (G93A) human Cu/Zn superoxide dismutase (SOD1) develop motor neuron pathology and clinical symptoms similar to those seen in patients with amyotrophic lateral sclerosis. Loss of motor neurons is most prominent in lumbar, followed by cervical cord and then brainstem. No significant cell death has been reported in motor cortex. The integrity of the cortical glutamate reuptake systems was evaluated using intracerebral microdialysis and western immunoblot assays for the glutamate transporters GLT-1, GLAST, and EAAC1. The basal extracellular fluid levels of aspartate, glutamate, glutamine, 3,4-dihydroxyphenylacetic acid, and 5-hydroxyindole-3-acetic acid were evaluated by HPLC. The extraction fraction of L-[3H]glutamate, corrected with [14C]mannitol, was also evaluated. GLT-1, EAAC1, and GLAST protein levels were determined by semiquantitative chemiluminescence immunoblot of proteins from membrane-enriched fractions. The relative optical density of film was translated into relative protein level by comparison with a standard control mouse. The SOD1 mutant mice demonstrated a significant (p < 0.05) increase in basal levels of extracellular aspartate and glutamate. In addition, when the glutamate extraction fraction was challenged with exogenous unlabeled glutamate (500 μM) by reversed microdialysis, the glutamate extraction fraction in the mutant SOD1 mice was decreased significantly from control levels. The SOD1 mutant mice demonstrated no difference in the cortical protein levels of the glutamate transporter subtypes. This study demonstrates that in areas of no visible pathology and no loss of glutamate transporter proteins, SOD1 mutant mice have elevated extracellular fluid aspartate and glutamate levels and a decreased capacity to clear glutamate from the extracellular space.
Amyotrophic lateral sclerosis (ALS) is a progressive disorder of the motor system usually leading to death within 2-5 years from the time of diagnosis. Although most cases of ALS are sporadic in origin, 10-15% are familial, and of these 15-20% possess a mutation in the gene that codes for the enzyme Cu/Zn superoxide dismutase (SOD1) (Siddique et al., 1991; Rosen et al., 1993). Transgenic mice expressing mutated human SOD1 demonstrate clinical symptoms and neuropathological findings similar to human ALS, whereas transgenic mice expressing normal human SOD1 are unaffected (Dal Canto and Gurney, 1994; Gurney et al., 1994; Ripps et al., 1995). Studies on SOD1 mutant mice suggest that motor neuron disease in these animals is not due to a reduction of SOD1 activity but rather a gain-of-function, possibly an enhancement in SOD1 capacity to generate reactive oxygen species (Yim et al., 1996; Bogdanov et al., 1998).
There is evidence for both oxidative stress and glutamate excitotoxicity in human ALS and in transgenic mice expressing mutant SOD1 (Meldrum and Garthwaite, 1990; Rothstein et al., 1992; Rosen et al., 1993; Bogdanov et al., 1998; Hall et al., 1998). These processes are not independent, but mutually reinforcing. Reactive oxygen species are known to inhibit the reuptake of glutamate, which would result in elevated glutamate levels (Volterra, 1994; Volterra et al., 1994; Trotti et al., 1996; Bogdanov et al., 1998), whereas elevated glutamate levels induce the production of reactive oxygen species (Reynolds and Hastings, 1995; Lancelot et al., 1998). Glutamate is the major excitatory transmitter in the mammalian CNS (Fonnum, 1984; Hansson and Ronnback, 1995). Its clearance from the extracellular space is regulated by Na+-dependent high-affinity excitatory amino acid transporters (Kanai et al., 1994). These reuptake systems are found in both neurons and glial cells (Kanai et al., 1995) and are critically important for the normal function of glutamatergic neurotransmission, as well as the maintenance of extracellular glutamate levels below potentially excitotoxic concentrations (Kanai et al., 1994).
Five different glutamate transporter subtypes [excitatory amino acid transporter one through five (EAAT1-EAAT5)] have been identified in humans (Pines et al., 1992; Arriza et al., 1994, 1995; Fairman et al., 1995; Robinson, 1998). Four (EAAT1-EAAT4) have been isolated in the CNS (Arriza et al., 1994; Fairman et al., 1995), and the fifth (EAAT5) is found in the retina (Arriza et al., 1997). Three of these isoforms correspond to transporters previously isolated in other species (Arriza et al., 1994). EAAT1 corresponds to the rat GLAST (Storck et al., 1992), EAAT2 to the rat GLT-1 (Pines et al., 1992), and EAAT3 to the rabbit and rat EAAC1 (Kanai and Hediger, 1992; Velaz-Faircloth et al., 1996). EAAC1 is localized to neuronal cell bodies throughout the brain, whereas EAAT4 is found mainly in cerebellar neurons (Fairman et al., 1995; Velaz-Faircloth et al., 1996). GLAST is expressed in astrocytes throughout the CNS and is most abundant in Bergmann glia in the cerebellum (Storck et al., 1992; Rothstein et al., 1994). GLT-1 is expressed in glial cells, primarily in astrocytes (Rothstein et al., 1994; Kanai et al., 1995). In addition, GLT-1 may also be expressed at low levels on the synaptic membrane of some glutamatergic neurons (Schmitt et al., 1996; Torp et al., 1997).
Postmortem tissue from ALS patients shows a significant (60-70%) protein loss of the glial glutamate transporter EAAT2 (GLT-1) chiefly in the motor cortex and spinal cord (Rothstein et al., 1995), primary targets of this disease. However, northern blot analysis shows no alterations in the mRNA levels of any of the glutamate transporter subtypes, even in tissue from patients demonstrating a significant decrease in EAAT2 protein (Bristol and Rothstein, 1996). Mice expressing a mutated human SOD1 gene (G85R) exhibit a 50% reduction of the glial glutamate transporter GLT-1 in the spinal cord, only at the end stage of the disease (Bruijn et al., 1997). Decreased glutamate transport has also been shown in spinal cord synaptosomes from transgenic mice expressing mutant (G93A) human SOD1 (Canton et al., 1998). Again, the reduction in glutamate transport in these animals is seen only in end-stage disease (Canton et al., 1998).
Mice expressing the G93A SOD1 mutation demonstrate increased brain production of hydroxyl free radicals (Bogdanov et al., 1998) and oxygen radical-induced lipid peroxidation in the spinal cord (Hall et al., 1998). The increased lipid peroxidation is seen in the spinal cord of 30-day-old animals. This may support a causative role for reactive oxygen species in the pathogenesis of motor neuron disease in SOD1 mutant animals because it occurs before clinical or pathological changes. A recent study using Xenopus oocytes expressing human GLT-1 and either normal or mutant human SOD1 demonstrated that oxidative stress, caused by hydrogen peroxide, resulted in inactivation of GLT-1 in the cells expressing mutant SOD1, but not in those expressing normal SOD1 (Trotti et al., 1999).
Cell culture, synaptosomal preparations, and postmortem studies, although often useful, are incapable of providing the necessary direct evidence of dysfunction in the intact brain. Intracerebral microdialysis offers a window into relevant operational parameters in a nonartificial environment. Intracerebral microdialysis can be used to sample, as well as deliver, small molecular weight compounds in the extracellular fluid (ECF) of the brains of awake, freely moving animals (Zetterström et al., 1983; Sharp et al., 1984; Rollema et al., 1989; Westerink, 1995). In this study, we evaluated the basal ECF glutamate level, its clearance, and the amount of the glutamate transporter protein subtypes in the motor cortex of B6SJL transgenic mice that express mutant (G93A) human SOD1 and demonstrate severe clinical symptoms. Results were compared with those obtained in companion B6SJL transgenic mice that express normal human SOD1 and in naive controls.
MATERIALS AND METHODS
Thirty-two mice varying in age from 120 to 165 days old were used in these studies: 14 controls, six animals expressing wild-type SOD1, and 12 animals expressing mutant (G93A) SOD1. Seven animals [three controls, one animal expressing wild-type SOD1, and three animals expressing mutant (G93A) SOD1] were used for neuropathologic studies, and the remaining animals were used for both intracerebral dialysis and western blot analysis of glutamate transporter proteins. The mice, obtained from our colony, were housed in a temperature- and light-controlled environment with free access to food and water. This colony was started from mice obtained from Jackson Laboratories (Bar Harbor, ME, U.S.A.) and originally produced by Gurney et al. (1994). Control SJL and mutant mice are purchased routinely from Jackson Laboratories to continue the colony. All animal protocols are approved by the MCP Hahnemann University Institutional Animal Care and Use Committee and conform to NIH guidelines.
All mice were genotyped using primers for human SOD1 as follows. A 1-1.5-cm piece of tail was clipped from each mouse and digested overnight at 50°C in buffer containing 50 mM Tris-HCl, pH 8.0, 5 mM EDTA, 0.5% sodium dodecyl sulfate, 200 mM NaCl, and 100 μg/ml proteinase K. An equal volume of a phenol (H2O-saturated)/chloroform/isoamyl alcohol solution was mixed in and centrifuged at 10,000 rpm for 10 min at 4°C. The top (aqueous) phase containing DNA was placed in a fresh centrifuge tube, and to it 1/10 volume of 3 M sodium acetate, pH 7, and 2× volume of ice-cold isopropanol were added. The tube was inverted to precipitate the DNA and then centrifuged at 10,000 rpm for 10 min at 4°C. The DNA pellet was cleaned in cold 70% ethanol and resuspended in 20 μl of sterile water. The presence of the SOD1 transgene was analyzed by amplification of the respective DNA samples. For the amplification reaction, 4 μl of DNA (1 μg/ml) was added to 95.4 μl of master mix containing (final concentrations) PCR buffer (50 mM KCl, 10 mM Tris-HCl, 0.1% Triton X-100; Promega), MgCl2 (2.5 mM; Promega), dNTP mix (1.0 mM; Promega), tetramethylammonium chloride (0.5 μM; Sigma), and forward and reverse primers (0.4 μM; Express Genetics) and denatured at 99°C for 5 min. Amplification was started by adding 0.6 μl (3 units) of Taq polymerase (Promega) and incubating in a thermal cycler (Hybaid). All samples were amplified for 35 cycles, each cycle consisting of denaturation at 94°C for 1 min, primer annealing at 60°C for 30 s, and extension at 72°C for 50 s. PCR products were visualized by electrophoresis on a 1.5% agarose gel stained with ethidium bromide. Primers were designed to amplify a portion of exon 2 of the human SOD1 gene and yield a 132-bp product (Rosen et al., 1993).
Glutamate studies were performed using stereotaxically placed microdialysis probes. On the night before surgery, the mice were allowed access to water only. The following morning, surgery was performed aseptically under tribromoethanol (250 mg/kg) anesthesia. The mice were positioned in a stereotaxic frame with mouse adaptor (David Kopf Instruments, Tujunga, CA, U.S.A.). A burr hole was drilled at 0.5 mm anterior to the bregma and 0.5 mm lateral to the midsagittal suture. The guide cannula (CMA/11 guide cannula, CMA/Microdialysis, Acton, MA, U.S.A.) was inserted stereotaxically at a 47° angle to the midsagittal plane to a 0.5-mm depth from the top of the brain. Two additional burr holes were drilled (anterior and posterior to the guide cannula) and skull screws inserted as anchors. The guide cannula was cemented to the skull with dental cement. The mouse was then removed from the stereotaxic frame and administered aspirin (20 mg/kg s.c.) as a postoperative analgesic. The animal was observed for a minimum of 6 h postoperatively to insure complete recovery from the anesthesia before being returned to the animal facility in a separate cage.
Following surgery, the animal was allowed a minimum recovery period of 18 h before the insertion of the dialysis probe. When inserted, the microdialysis probe membrane extends 3 mm beyond the end of the guide cannula and is located in the sensorimotor cortex (Fig. 1A). CMA/11 microdialysis probes (CMA/Microdialysis) have a molecular mass cutoff of 6 kDa, a membrane diameter of 0.25 mm, and a membrane length of 3 mm. Before their use in an animal experiment, the microdialysis probes were calibrated in vitro at a constant temperature of 37°C. The recoveries for L-[3H]glutamate (17.8 Ci/mmol) and [14C]mannitol (51.5 mCi/mmol) (New England Nuclear, Boston, MA, U.S.A.) were determined at a flow of 1 μl/min.
Throughout the course of the experiment, the animal was able to move freely about its cage. Artificial cerebrospinal fluid (25 mM NaHCO3, 122 mM NaCl, 1.3 mM CaCl2, 1.2 mM MgSO4, 3 mM KCl, and 0.4 mM KH2PO4), pH 7.35, was perfused through the probe at a flow rate of 1 μl/min and the effluent collected into small microcentrifuge tubes. Sampling was started 3 h after the insertion of the microdialysis probe. Samples were collected at 20- or 30-min intervals and analyzed immediately by HPLC for biogenic amines and amino acid levels.
Amino acids (aspartate, glutamate, and glutamine) were quantified using precolumn derivatization of samples with o-phthaldialdehyde. The o-phthaldialdehyde-derivatized amino acids were analyzed immediately using HPLC with fluorometric detection. HPLC of biogenic amines was performed on an octadecylsilane (C18) column (Microsorb Short-One, Rainin Instrument Co., Woburn, MA, U.S.A.). Coulometric detection (Coulochem II 5200A, ESA, Inc., Bedford, MA, U.S.A.) was accomplished using sequential oxidation and reduction with the guard cell at a potential of -0.4 V, electrode no. 1 at +0.4 V, and electrode no. 2 at -0.35 V. Statistical significance between groups was determined by analysis of variance. The groups were considered significantly different if p < 0.05.
For neuropathologic studies, the animals were killed by intraperitoneal injection of Beuthanasia-D (solution of sodium pentobarbital and sodium phenytoin; Schering Corp., Kenilworth, NJ, U.S.A.) at a dose containing 150 mg/kg sodium pentobarbital. The animals were perfused transcardially with 4% paraformaldehyde in 0.13 M sodium phosphate buffer. The brain was postfixed for 1 day, dehydrated in incremental concentrations of ethanol, and embedded in paraffin. Routine histopathology with hematoxylin and eosin staining was carried out on transverse sections through the brain, brainstem, and spinal cord. In addition to routine histochemistry, immunostaining with antibodies to glial fibrillary acidic protein was performed to examine whether gliosis was present.
At the completion of the experiment, in the animals that underwent intracerebral dialysis, the brain was removed, the placement of the dialysis probe was verified, and the sensori-motor cortex was quickly dissected out in ice-cold phosphate-buffered saline for western blot analysis of transporter proteins. The sensorimotor cortex was homogenized in 10 mM Tris, pH 7.4, 1 mM EDTA, 0.32 M sucrose, 1 mM sodium orthovanadate, 0.5 mM phenylmethylsulfonyl fluoride, and 5 mM dithiothreitol (HB buffer), and centrifuged to collect a membrane-enriched pellet. The final pellet was resuspended in 1 mM EDTA, 50 mM Tris, pH 7.4, and combined with sample buffer (62.5 mM Tris, pH 6.8, 2.6% sodium dodecyl sulfate, 2.5% β-mercaptoethanol, 8 M urea, and 0.03% bromphenol blue, final concentrations). Protein concentrations were assayed by the method of Lowry et al. (1951) on homogenates before mixing with sample buffer. Samples from each specimen containing either 2 or 20 μg of total protein were separated on 7.5% polyacrylamide gels, transferred to nitrocellulose, and immunoblotted with antibodies to the glutamate transporters GLT-1 (Affinity Bioreagents, Golden, CO, U.S.A.) or GLAST and EAAC1 (Alpha Diagnostic International, San Antonio, TX, U.S.A.). Labeling was visualized with chemiluminescence (Renaissance, Du Pont) and recorded on film at two or more exposure times. Films were scanned using a Hewlett-Packard scanner and analyzed using Image Pro 3.0 software (Media Cybernetics, Silver Spring, MD, U.S.A.). Equivalent protein loading was checked by reprobing each blot with an antibody to connexin 43 (Zymed Laboratories, San Francisco, CA, U.S.A.), a prominent membrane protein. Gels demonstrating unequal protein loading were discarded.
For quantitative purposes, every gel included three lanes loaded with 1, 2, and 5 μg of total protein from a control mouse chosen as a standard. These lanes were used to create a predictive linear regression of the relationship between optical density of an immunolabeled band and the amount of total protein loaded on the gel. By using the Systat statistical package (SPSS), the integrated optical density of the immunoreactive GLT-1 band(s) was converted into a number relative to the amount of GLT-1 in the standard. The GLT-1 contents of transgenic and control mice were then compared by their relative amounts.
The mice in our colony exhibited a phenotype consistent with animals possessing 25 copies of the mutant SOD1 (G93A) transgene with onset of clinical symptoms at 90-100 days of age (Chiu et al., 1995). At this time, there is wasting, abnormal splay of the hindlimbs, and tremor. The disease progresses over subsequent weeks, resulting in death at 150-170 days of age. The SOD1 mutant mice used in this study demonstrated hindlimb paralysis between 120 and 160 days of age. At all times, these animals were able to eat, drink, and move around the cage using their front limbs, and were never allowed to reach the stage where they were unable to right themselves when placed on their side. Histopathologic examination of routine hematoxylin stains and glial fibrillary acidic protein demonstrated severe loss of neurons in the lumbar and cervical spinal cords along with gliosis in SOD1 mutant mice similar to changes reported previously (Dal Canto and Gurney, 1994). Although neurons were well preserved in the brainstem region of the mutants, there was gliosis extending to the level of the colliculus. The cortex of these mice demonstrated no pathologic change (Fig. 1B and C) or gliosis. The average age at which the intracerebral microdialysis studies were performed in the SOD1 mutant animals was 145.5 ± 3.4 days. The control and wild-type SOD1 animals were age-matched to the mutants.
The basal cortical dialysate concentrations of aspartate, glutamate, glutamine, 3,4-dihydroxyphenylacetic acid (DOPAC), and 5-hydroxyindole-3-acetic acid (HIAA), as well as the basal extraction fraction for glutamate corrected for mannitol (EFL-glu) for the animals in this study, are tabulated in Table 1. There was no significant difference in the dialysate levels of aspartate, glutamate, glutamine, DOPAC, and 5-HIAA between controls and wild-type SOD1 animals. However, the dialysate levels of aspartate and glutamate were increased significantly (p < 0.05) in the SOD1 mutant animals as compared with controls or wild-type SOD1 mice (Fig. 2).
|Controls (n = 11)||Wild-type (n = 5)||Mutant (n = 10)|
|Age (days)||143.3 ± 4.9||141.2 ± 6.9||145.5 ± 3.4|
|Aspartate (μM)||0.752 ± 0.11||0.619 ± 0.10||1.137 ± 0.17a|
|Glutamate (μM)||1.166 ± 0.12||1.338 ± 0.19||2.109 ± 0.18b|
|Glutamine (μM)||42.196 ± 2.24||41.090 ± 6.40||40.382 ± 2.79|
|5-HIAA (μM)||0.093 ± 0.02||0.094 ± 0.01||0.106 ± 0.01|
|DOPAC (μM)||0.065 ± 0.01||0.076 ± 0.02||0.096 ± 0.01|
|EFL-glu (ratio)||0.131 ± 0.01||0.162 ± 0.01||0.152 ± 0.01|
The microdialysis probes demonstrated no significant difference (p > 0.05) between the in vitro recoveries of L-[3H]glutamate (0.64 ± 0.01) and [14C]mannitol (0.65 ± 0.01). The cortical EFL-glu ranged from 10 to 19% with a mean of 14.6%. There was no significant (p > 0.05) difference in EFL-glu among controls, wild-type SOD1, and mutant SOD1 animals (Table 1).
When the glutamate extraction was challenged by the addition of exogenous unlabeled glutamate to the dialysate, EFL-glu demonstrates a decrease that is proportional to the unlabeled glutamate concentration and is dependent on the capacity of the brain structure to clear glutamate from the extracellular space (Alexander et al., 1997) (Fig. 3). When the reuptake system of control mice was challenged by the addition to the dialysate of 500 μM L-glutamate, the EFL-glu demonstrated a 22% decrease. In contrast, the severely affected SOD1 mutant animals demonstrated a significantly greater decrease (42%) in EFL-glu (p < 0.01) (Fig. 4).
Immunoblots of GLT-1 in our preparations label two bands: one at 70 kDa (the GLT-1 monomer) and one at 140 kDa (presumably a dimer form). For quantification, the integrated optical density of the two bands was summed to give the total amount of GLT-1 protein for each subject. Figure 5 shows an example of a western blot comparing GLT-1 levels in samples from four severely affected transgenic mice and two age-matched controls. The standards occupy the first three lanes and contain 0.5, 2.0, and 5.0 μg of total protein from a control mouse (this mouse was used in all GLT-1 immunoblots). Each additional lane contains 2 μg of total protein. The amount of GLT-1 in each experimental lane was determined by comparison with the linear regression line created from the standards. The relative levels of GLT-1 protein in SOD1 mutants and controls is illustrated in Fig. 6. There was no significant (p > 0.05) difference in the amount of GLT-1 protein between groups. There was also no significant difference in the total amount of GLAST or EAAC1 protein between controls and SOD1 mutant animals (data not shown).
This study demonstrates that the ECF levels of the excitatory amino acids glutamate and aspartate are significantly (p < 0.05) elevated in the sensorimotor cortex of severely affected transgenic mice expressing mutant (G93A) human SOD1 as compared with age-matched naive animals or transgenic mice expressing normal human SOD1. The ECF elevation of excitatory amino acids presumably preceded pathology, because these animals demonstrated no evidence of neurodegeneration or inflammation and no gliosis in the sensorimotor cortex, although it is uncertain whether this animal model would demonstrate cortical pathology given sufficient survival time.
Several possible explanations could account for elevated levels of glutamate in the ECF of SOD1 mutant mice. We do not feel that the increase in cortical extra-cellular glutamate in the SOD1 mutant mice was caused by hypoxia due to inadequate ventilation. The severely affected animals were not moribund and unable to ventilate properly. They demonstrate hindlimb paralysis, but were able to eat, drink, and move around the cage using their front limbs and were never allowed to reach the stage where they were unable to right themselves when placed on their side. In addition, we have recently presented data that show increased cortical extracellular glutamate in asymptomatic mice as young as 60 days old (Alexander et al., 1999). The glutamate concentration in the ECF is dependent on its rate of release and its rate of removal by metabolism and reuptake. There is no evidence of the extracellular metabolism of the excitatory amino acids (Robinson, 1998); the enzymes involved in glutamate metabolism are located intracellularly in neurons and glia (Schousboe, 1981; Peng et al., 1993). An increase in the release of glutamate and/or a decrease in its reuptake are the likely causes of the observed elevated extracellular glutamate seen in the SOD1 mutant animals.
That SOD1 mutant mice have impaired reuptake of glutamate is supported by our intracerebral dialysis studies on glutamate extraction. When the glutamate extraction was challenged by the addition of 500 μM glutamate to the dialysate, the SOD1 mutant mice demonstrated a significantly greater decrease (p < 0.05) in the EFL-glu as compared with the controls or the transgenic mice expressing normal human SOD1. This indicates an impairment of glutamate reuptake that is not due to a reduction of transporters, because the level of the glutamate transporter protein subtypes in the SOD1 mutants was unchanged from control values.
Recent studies show that glutamate transport is affected by oxidative stress (Trotti et al., 1997b, 1999). Increased reactive oxygen species production can cause oxidation of glutamate transporters, resulting in a reduction of glutamate reuptake (Trotti et al., 1997b, 1999). All of the glutamate transporter subtypes demonstrate impaired glutamate reuptake capacity when exposed to reactive oxygen species (Trotti et al., 1997a). However, due to factors such as their physical location or the number of reactive sulfhydryls present in their structure (GLT-1 has nine cysteines, EAAC1 has six, and GLAST has three) (Arriza et al., 1997; Trotti et al., 1997a,b), some transporter subtypes could be more susceptible to oxidative stress than others. A recent study using Xenopus oocytes expressing human GLT-1 and either normal or mutant human SOD1 demonstrated that oxidative stress, caused by hydrogen peroxide, resulted in inactivation of GLT-1 in the cells expressing mutant (A4V) SOD1, but not in those expressing normal SOD1 (Trotti et al., 1999). The reuptake inhibition was specific for GLT-1, because glutamate reuptake in cells expressing EAAC1 and A4V mutant SOD1 were unaffected by the addition of hydrogen peroxide (Trotti et al., 1999). These investigators demonstrated that the oxidation target site was in the carboxyl terminus of GLT-1, where GLT-1 is rich in amino acid residues susceptible to oxidation, whereas EAAC1 is not (Trotti et al., 1999).
Mice expressing the G93A SOD1 mutation have been shown to have increased brain production of hydroxyl free radicals (Bogdanov et al., 1998), exposing the CNS of these animals to increased oxidative stress. The fact that GLT-1 has increased susceptibility to reactive oxygen species (Trotti et al., 1999) would render some areas of the CNS more vulnerable to oxidative stress due to their mix of glutamate transporter subtypes. For instance, the motor neurons in the spinal cord depend mainly on GLT-1 to clear glutamate from the ECF because there is only moderate expression of EAAC1 and the low GLAST expression is dorsal, not ventral (Sutherland et al., 1996; Furuta et al., 1997; Shibata et al., 1997).
These studies are the first in vivo demonstration of altered glutamate clearance capacity in the CNS of G93A SOD1 transgenic mice and how these changes correlate with the level of the glutamate transporter protein subtypes. Our data show elevated ECF glutamate levels in the sensorimotor cortex of SOD1 mutant mice. In addition, when the reuptake system of SOD1 mutant mice was challenged by addition of 500 μM L-glutamate to the dialysate, the EFL-glu demonstrated a decrease that was almost twice that shown by controls. This reduction of glutamate reuptake capacity is present even though the glutamate transporter protein levels were unchanged from control levels. These findings are consistent with the hypothesis that the increased oxidative stress previously reported in these mice (Bogdanov et al., 1998) causes oxidation of glutamate transporter proteins, resulting in a decreased capacity to clear extracellular glutamate. Further studies will be needed at different ages and in different areas of the brain to delineate the role of decreased glutamate reuptake capacity in the mechanism of motor neuron disease seen in this model of ALS.
This study was supported by a grant from the Amyotrophic Lateral Sclerosis Association.
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