Sage Journals: Discover world-class research

Abstract

Although degeneration of lower motor neurons is the most striking abnormality in amyotrophic lateral sclerosis (ALS), more subtle alterations may occur in the brain. Mutations in copper/zinc superoxide dismutase (Cu/Zn-SOD) are responsible for some cases of inherited ALS, and expression of mutant Cu/Zn-SOD in transgenic mice results in progressive motor neuron loss and a clinical phenotype similar to that of ALS patients. It is now reported that Cu/Zn-SOD mutant mice exhibit increased vulnerability to focal ischemic brain injury after transient occlusion of the middle cerebral artery. Levels of glucose and glutamate transport in cerebral cortex synaptic terminals were markedly decreased, and levels of membrane lipid peroxidation were increased in Cu/Zn-SOD mutant mice compared to nontransgenic mice. These findings demonstrate that mutant Cu/Zn-SOD may endanger brain neurons by a mechanism involving impairment of glucose and glutamate transporters. Moreover, our data demonstrate a direct adverse effect of the mutant enzyme on synaptic function.

Keywords

Amyotrophic lateral sclerosis Excitotoxicity Focal ischemia Lipid peroxidation Transgenic

Amyotrophic lateral sclerosis (ALS) is a progressive and always fatal neurodegenerative disorder that involves death of motor neurons in the spinal cord. Analyses of cerebrospinal fluid and postmortem spinal cord tissue from ALS patients have demonstrated increased levels of oxidative stress (lipid peroxidation and protein oxidation), (Beal et al., 1997; Ferrante et al., 1997a; Pedersen et al., 1998), increased levels of extracellular glutamate, and reduced levels of glutamate transporters (see Rothstein, 1995 for review), in ALS patients. Although the cause of most cases of ALS is unknown, some cases result from heritable mutations in Cu/Zn-SOD (Gurney et al., 1996). The Cu/Zn-SOD mutations do not compromise the normal dismutase activity of the enzyme but instead result in gain of an adverse property of the mutant protein which may involve increased peroxidase activity resulting in hydroxyl radical production (Wiedau-Pazos et al., 1996; Yim et al., 1997). Expression of ALS-linked Cu/Zn-SOD mutations in transgenic mice results in progressive degeneration of spinal cord motor neurons and associated paralysis (Gurney et al., 1994; Wong et al., 1995). Studies of spinal cord motor neurons from the ALS mice in cell culture and in vivo have provided evidence that the Cu/Zn-SOD mutations result in increased levels of oxidative stress and increased vulnerability to excitotoxicity (Ferrante et al., 1997b; Siklos et al., 1998; Roy et al., 1998; Kruman et al., 1999). The pathogenic action of Cu/Zn-SOD mutations in familial ALS does not result from a loss of dismutase function of the enzyme, but rather from gain of an adverse property of the mutant protein. Thus, there is no correlation between enzyme activity and disease severity in ALS patients and Cu/Zn-SOD mutant mice, and enzyme activity appears to be increased consistent with the level of enzyme overexpression (Gurney et al., 1994; Ripps et al., 1995; Wong et al., 1995). Moreover, Cu/Zn-SOD knockout mice are viable, live well beyond 1 year of age, and fail to develop motor neuron disease (Reaume et al., 1996). The specific alteration in the mutant enzyme has not been established but might result from enhanced hydroxyl radical production compared with the wild-type protein (Wiedau-Pazos et al., 1996; Yim et al., 1997).

Oxidative stress, impaired energy metabolism, and overactivation of glutamate receptors are increasingly implicated in the pathogenesis of neurodegenerative disorders ranging from Alzheimer's and Parkinson's diseases (Mattson, 1997; Jenner and Olanow, 1998) to stroke (Chan, 1996). Because glutamate receptors are concentrated in postsynaptic terminals, the excitotoxic process (be it apoptosis or necrosis) is initiated in synaptic compartments and propagates to the cell body (Mattson et al., 1998; Duan et al., 1999). Oxidative stress and lipid peroxidation can impair the function of glutamate and glucose transporters (Keller et al., 1997a; Mark et al., 1997a; Blanc et al., 1998) and ion-motive ATPases (Mark et al., 1995, 1997b) in neuronal cultures and synaptosome preparations. Impairment of these transporters results in increased levels of extracellular glutamate, ATP depletion and membrane depolarization, and excessive influx of calcium through N-methyl-d-aspartate receptors and voltage-dependent channels.

Metabolic and oxidative alterations in neurodegenerative disorders are often not limited to the neuronal populations that ultimately succumb (see Govoni et al., 1996; Connolly, 1998; Schapira et al., 1998 for review). A few studies suggest that abnormalities in ALS are not limited to the spinal cord. For example, positron emission tomography studies of cerebral glucose utilization have documented reduced levels of glucose uptake throughout many regions of cerebral cortex of ALS patients (Ludolph et al., 1992). Levels of glutamate transport proteins are decreased in cerebral cortex of ALS patients (Rothstein et al., 1995), and ALS patients exhibit abnormalities in carbohydrate metabolism (Murai et al., 1983). It is not known whether such abnormalities precede motor neuron degeneration or are secondary phenomena. In the present study we directly tested the hypothesis that abnormalities are present in the brains of Cu/Zn-SOD transgenic mice before onset of motor dysfunction and that such alterations place the neurons “at risk.” We report that synaptic glucose and glutamate transport are impaired in cerebral cortex of the ALS mice and that these abnormalities are associated with increased vulnerability to ischemic injury.

MATERIALS AND METHODS

Mice and transient focal cerebral ischemia protocol

Transgenic mice overexpressing human Cu/Zn-SOD with the ALS-linked G93A mutation (TgN[SODl-G93A]1Gur) were purchased from Jackson Laboratories (Bar Harbor, ME, U.S.A.). Mice were maintained on a B6/SJL background, and transgenic and nontransgenic offspring of crosses of heterozygous mice were used for experiments. Mice were maintained on a 12-hour light: 12-hour dark cycle and were provided free access to food and water. Cu/Zn-SOD mutant mice used in the present study developed progressive lower motor neuron degeneration and associated paralysis beginning from 5 to 7 months of age. All experiments were performed on 3-month-old male mice. The protocol for performing focal cerebral ischemia using a middle cerebral artery occlusion-reperfusion method was similar to that used in our previous studies (Bruce et al., 1996; Keller et al., 1998) and was adapted from Huang et al. (1994). Briefly, mice were anesthetized with 350 mg/kg chloral hydrate. Thermistor probes were inserted into the rectum and temporalis muscles to monitor body and brain temperature, which were maintained at 36 to 37°C by external warming. Blood samples were taken at baseline (10 minutes before occlusion), during ischemia (30 to 35 minutes after thread placement), and 10 minutes after reperfusion for measurements of blood gases and pH level. The left common carotid artery was exposed and the occipital, superior thyroidal, maxillary, and lingual branches of the external carotid artery were coagulated; the pterygopalatine artery was ligated. An incision was made in the external carotid artery and a polylysine-coated suture was advanced into the middle cerebral artery. The thread was left in place for 60 minutes and then removed to allow reperfusion. After surgery mice were returned to their cages and given free access to food and water. Neurologic deficits were assessed 22 hours after ischemia based on a scale from 0 (no deficits) to 3 (severe deficits) as described previously (Huang et al., 1994; Endres et al., 1999). At 24 hours after ischemia the mice were euthanized by anesthesia overdose, and coronal brain sections (1 mm thick) were stained with 1% triphenyltetrazolium chloride. Images of the stained brain slices were captured using a digital camera, and the infarct volume was quantified as described previously (Keller et al., 1998).

Synaptosome preparation and experimental treatment

Eight wild-type and 12 PS 1 mutant mice were used for these studies. For each synaptosome preparation, cerebral hemispheres from 2 mice were pooled and homogenized in a solution containing 0.32 mol/L sucrose, 4 μLg/mL pepstatin, 5 μLg/mL aprotinin, 20 μLg/mL trypsin inhibitor, 4 μLg/mL leupeptin, 0.2 mmol/L phenyl-methylsulfonyl fluoride, 2 mmol/L EDTA, 2 mmol/L EGTA, and 20 mmol/L Hepes. Synaptosomes were prepared using a sucrose density gradient protocol described in our previous studies (Keller et al., 1997; Begley et al., 1999). Protein concentrations in synaptosomal preparations were determined (Pierce BCA kit), and the synaptosomes were diluted in Locke's buffer (NaCl, 154 mmol/L; KCl, 5.6 mmol/L; CaCl₂, 2.3 mmol/L; MgCl₂, 1.0 mmol/L; NaHCO₃, 3.6 mmol/L; glucose, 5 mmol/L; Hepes, 5 mmol/L; pH 7.2) to a final concentration of 200 μLg protein/mL. Synaptosomes were then aliquoted into 1.5-mL Eppendorf tubes for glucose and glutamate transport assays or into wells of 96-well plates for measurement of lipid peroxidation. Synaptosomes were either left untreated or were exposed to 10 μmol/L FeSO₄.

Measurement of glucose and glutamate transport and lipid peroxidation

The methods for quantifying glutamate and glucose transport in synaptosomes have been described previously (Keller et al., 1997). For the glutamate uptake assay, synaptosomes (200 μLg/tube) were incubated for 7 minutes with [³H]-glutamate (0.1 μCi/mL). The synaptosomes were then pelleted, washed three times in Locke's buffer, and lysed in 200 μL of solution of 1% sodium dodecyl sulfate in phosphate-buffered saline. The lysate was placed in scintillation vials containing Scintiverse, and radioactivity was counted in a Packard 2500TR liquid scintillation counter. For the glucose uptake assay, synaptosomes (200 μg/tube) were subjected to experimental treatments and then washed three times in glucose-free Locke's buffer, and the assay was started by the addition of 1.5 μCi of [³H]-2-deoxyglucose. Seven minutes later the assay was stopped by pelleting the synaptosomes, washing twice with glucose-free Locke's solution, and lysing the synaptosomes in 200 μL of a 1% sodium dodecyl sulfate-phosphate-buffered saline solution. The lysate was placed in scintillation vials containing Scintiverse, and radioactivity was counted in a Packard 2500TR liquid scintillation counter. Results were expressed as counts per minute per milligram of protein. The rates of glucose and glutamate uptake by synaptosomes remained constant under basal conditions throughout the time course of experiments (up to 4 hours). In such assays of glucose and glutamate transport there is often considerable interassay variability in terms of counts per minute of synaptosome-associated radioactivity, whereas intraassay variability is quite low. Because we always included control conditions within an experiment, the experimental values were compared directly to the control value within the experiment. Levels of membrane lipid peroxidation were assessed using a thiobarbituric acid-reactive substances fluorescence-based method described previously (Keller et al., 1998). Values were expressed as a percentage of the value in untreated synaptosomes from wild-type mice.

RESULTS AND DISCUSSION

Previous studies have shown that in the same transgenic line of mice used in the present study, mutant Cu/Zn-SOD is expressed in cells throughout the nervous system, although levels of expression are somewhat lower in the brain as compared to the spinal cord (Liu et al., 1998). Our preliminary studies confirmed expression of the transgene in cerebral cortical tissue, wherein levels of overall Cu/Zn-SOD enzyme activity were increased approximately 2- to 3-fold (data not shown). In order to determine whether mutant Cu/Zn-SOD affects the vulnerability of cortical neurons to injury induced by metabolic/excitotoxic insults we subjected nontransgenic (wild-type) mice (n = 10) and Cu/Zn-SOD mutant mice (n = 10) to transient middle cerebral artery occlusion, a model of focal ischemic stroke. There were no significant differences in physiologic parameters (mean arterial pressure; blood o₂, co₂, pH, and glucose; cerebral blood flow) between wild-type and mutant mice at baseline, during ischemia, or after reperfusion (Table 1). Infarct volume, measured 24 hours after ischemia, was significantly increased in Cu/Zn-SOD mutant mice compared to wild-type mice (Fig. 1). The increased brain damage in the mutant mice was associated with increased behavioral deficits (Fig. 1). The increased vulnerability to focal ischemia—reperfusion injury in Cu/Zn-SOD mutant mice is apparently attributable to the mutation in the enzyme rather than to overexpression of the enzyme, because previous studies using the same ischemia-reperfusion model have shown that transgenic mice overexpressing wild-type Cu/Zn-SOD exhibit decreased infarct size after focal ischemia (Yang et al., 1994). We observed no differences in cerebral vascular anatomy, and because cerebral blood flow was similar in mutant and nontransgenic mice (before, during, and after ischemia), indicating that mutant Cu/Zn-SOD did not affect vascular structure or physiology.

FIG. 1.

Increased infarct size and behavioral deficits after transient focal cerebral ischemia in copper/zinc superoxide dismutase (Cu/Zn-SOD) mutant mice. Values are the mean and SD (n = 10 mice). *P < 0.01 compared to value for wild-type mice (ANOVA with Scheffe's posthoc tests).

Ischemic neuronal injury involves ATP depletion, oxidative stress, and overactivation of glutamate receptors which results in calcium overload, mitochondrial dysfunction, and either apoptosis or necrosis (see Chan, 1996; Mattson et al., 1999 for review). To elucidate the mechanism(s) responsible for the endangering action of mutant Cu/Zn-SOD toward cortical neurons, we studied cortical synaptosomes from wild-type and Cu/Zn-SOD mutant mice. It was previously reported that levels of membrane lipid peroxidation are increased in spinal cords of ALS patients and Cu/Zn-SOD mutant mice (Hall et al., 1998; Pedersen et al., 1998). Measurement of relative levels of membrane lipid peroxidation, using a thiobarbituric acid-reactive substances assay, revealed a small but significant increase in basal levels of lipid peroxidation in synaptosomes from transgenic mice compared to wild-type mice (Fig. 2A). The magnitude of the increase in lipid peroxidation induced by Fe²⁺ was also significantly increased in synaptosomes from the transgenic mice, suggesting an increased sensitivity to oxidative stress.

FIG. 2.

Levels of membrane lipid peroxidation are increased, and glucose and glutamate transport are decreased in cortical synaptosomes from copper/zinc superoxide dismutase (Cu/Zn-SOD) mutant mice. (A) Thiobarbituric acid-reactive substances (TBARS) fluorescence was quantified in synaptosomes from wild-type and Cu/Zn-SOD mutant mice under basal conditions and 4 hours after exposure to 10 μmol/L FeSO₄. Values are the mean and SD of determinations made in 4 to 6 synaptosome preparations. *P < 0.05, **P < 0.01 compared to value for wild-type mice. (B) Levels of glucose and glutamate uptake were quantified in synaptosomes from wild-type and Cu/Zn-SOD mice. Values are the mean and SD of determinations made in 4 to 6 synaptosome preparations. *P < 0.01, **P < 0.001 compared to value for wild-type mice.

TABLE 1.

Physiologic parameters in nontransgenic (wild-type) mice and Cu/Zn-SOD mutant transgenic mice

	Beforeischemia	Duringischemia	Afterischemia
Cerebral blood flow (% baseline)
Wild-type	100 ± 4	21 ± 5	95 ± 6
Cu/ZnSODMut	100 ± 6	25 ± 6	99 ± 5
Blood pressure (mm Hg)
Wild-type	89.1 ± 9.2	88.6 ± 10.4	91.7 ± 8.9
Cu/ZnSODMut	87.5 ± 10.2	86.9 ± 9.9	89.9 ± 10.6
pH
Wild-type	7.36 ± 0.05	7.34 ± 0.04	7.33 ± 0.04
Cu/ZnSODMut	7.35 ± 0.03	7.34 ± 0.05	7.34 ± 0.03
Po₂ (mm Hg)
Wild-type	92.4 ± 3.8	93.2 ± 4.2	93.8 ± 4.9
Cu/ZnSODMut	92.5 ± 4.3	91.2 ± 4.4	92.5 ± 5.1
Pco₂ (mm Hg)
Wild-type	47.5 ± 3.7	48.3 ± 3.9	49.8 ± 5.1
Cu/ZnSODMut	50.4 ± 4.3	50.1 ± 5.0	49.9 ± 4.5

Measurements were made 10 minutes before occlusion, during ischemia (10 minutes after occlusion), and 10 minutes after reperfusion (except for cerebral blood flow which was measured 30 minutes after reperfusion).

We had previously shown that glucose and glutamate transport in cortical synaptosomes are impaired after exposure to insults that induce membrane lipid peroxidation (Keller et al., 1997a, b ). Because impairment of glucose and glutamate transport would be expected to render neurons vulnerable to excitotoxicity, we measured levels of glucose and glutamate transport in synaptosomes from nontransgenic and transgenic mice. There was a striking 70% reduction in the level of glucose transport in synaptosomes from Cu/Zn-SOD mutant mice compared to wild-type mice (Fig. 2B). The level of glutamate transport was also significantly reduced by approximately 40% in synaptosomes from Cu/Zn-SOD mutant mice (Fig. 2B). In light of these findings and because we observed no differences in cerebral vascular anatomy and because cerebral blood flow was similar in mutant and nontransgenic mice (before, during, and after ischemia), we conclude that the increased infarct size after focal ischemia-reperfusion in the the Cu/Zn-SOD mutant mice results from an adverse effect of the mutant enzyme in synaptic terminals of neurons. However, a contribution of indirect effects of the mutant enzyme on the vasculature or glial cells cannot be ruled out.

Previous studies have provided evidence that levels of oxidative stress are increased in cerebral cortex of ALS patients (Ferrante et al., 1997a), and that levels of lipid peroxidation and protein nitration are increased in cerebral cortical tissue from transgenic mice expressing an ALS-linked Cu/Zn-SOD mutation (Ferrante et al., 1997b). The latter studies involved analyses of brain tissue taken from end-stage ALS patients and Cu/Zn-SOD mutant mice. Our studies, performed on brain tissue taken several months before the onset of symptomatic motor dysfunction, suggest that cortical neurons are subjected to increased oxidative stress during the early stages of the disease process. Measurements of lipid peroxidation in spinal cord tissue of Cu/Zn-SOD mutant mice (Hall et al., 1998) and motor neurons in cultures established from embryonic Cu/Zn-SOD mutant mice (Kruman et al., 1999) also indicate that lipid peroxidation occurs before motor neuron degeneration. We found striking deficits in glucose and glutamate transport in cortical synaptosomes from the Cu/Zn-SOD mutant mice suggesting that the neurons are subjected to metabolic and excitotoxic stress. Although the mechanism whereby mutant Cu/Zn-SOD causes impaired glucose and glutamate transport is unknown, it might result from increased oxidative stress. Studies of hippocampal and cortical cell cultures and synaptosomes (Keller et al., 1997a, b ; Mark et al., 1997a, b ; Blanc et al., 1998) and motor neuron-like cells (Pedersen et al., 1999) have shown that membrane lipid peroxidation results in impairment of glucose and glutamate transporters. In addition, mutant Cu/Zn-SOD was reported to inactivate the glutamate transporter GLT-1 (Trotti et al., 1999). The beneficial effect of antioxidants, such as vitamin E that act by suppressing lipid peroxidation in ALS mice (Gurney et al., 1996), is consistent with a role for oxidative impairment of glucose and glutamate transport in the pathogenesis of ALS. Our findings may also provide an explanation for the widespread decrease in glucose utilization (Ludolph et al., 1992) and the decreased levels of glutamate transporter protein GLT-1 (Rothstein et al., 1995) in cerebral cortex of ALS patients.

The demonstration of impaired glucose and glutamate transport in cortical synaptosomes from Cu/Zn-SOD mutant mice provides the first direct evidence for synaptic dysfunction in ALS. Our findings support the excitotoxicity hypothesis of ALS in that impaired glucose and glutamate transport in synaptic terminals would predispose neurons to excitotoxicity.

Footnotes

Acknowledgments

The authors thank W. A. Pedersen and H. Zhu for technical assistance.

Abbreviations used

References

Beal

Ferrante

Browne

Matthews

Kowall

Brown

Jr (1997) Increased 3-nitrotyrosine in both sporadic and familial amyotrophic lateral sclerosis. Ann Neurol 42:644–654

Begley

Duan

Duff

Mattson

(1999) Altered calcium homeostasis and mitochondrial dysfunction in cortical synaptic compartments of presenilin-1 mutant mice. J Neurochem 72:1030–1039

Blanc

Keller

Fernandez

Mattson

(1998) 4-hydroxynonenal, a lipid peroxidation product, impairs glutamate transport in cortical astrocytes. Glia 22:149–160

Bruce

Boling

Kindy

Peschon

Kraemer

Carpenter

Holtsberg

Mattson

(1996) Altered neuronal and microglial responses to brain injury in mice lacking TNF receptors. Nat Med 2:788–794

Chan

(1996) Role of oxidants in ischemic brain damage. Stroke 27:1124–1129

Connolly

(1998) Fibroblast models of neurological disorders: fluorescence measurement studies. Trends Pharmacol Sci 19:171–177

Duan

Rangnekar

Mattson

(1999) Prostate apoptosis response-4 production in synaptic compartments following apoptotic and excitotoxic insults: evidence for a pivotal role in mitochondrial dysfunction and neuronal degeneration. J Neurochem 72:2312–2322

Endres

Fink

Zhu

Stagliano

Bondada

Geddes

Azuma

Mattson

Kwiatkowski

Moskowitz

(1999) Neuroprotective effects of gelsolin during murine stroke. J Clin Invest 103:347–354

Ferrante

Browne

Shinobu

Bowling

Baik

MacGarvey

Kowall

Brown

Jr Beal

(1997a) Evidence of increased oxidative damage in both sporadic and familial amyotrophic lateral sclerosis. J Neurochem 69:2064–2074

10.

Ferrante

Shinobu

Schulz

Matthews

Thomas

Kowall

Gurney

Beal

(1997b) Increased 3-nitrotyrosine and oxidative damage in mice with a human copper/zinc superoxide dismutase mutation. Ann Neurol 42:326–334

11.

Govoni

Gasparini

Racchi

Trabucchi

(1996) Peripheral cells as an investigational tool for Alzheimer's disease. Life Sci 59:461–468

12.

Gurney

Cutting

Zhai

Doble

Taylor

Andrus

Hall

(1996) Benefit of vitamin E, riluzole, and gabapentin in a transgenic model of familial amyotrophic lateral sclerosis. Ann Neurol 39:147–157

13.

Gurney

Chiu

Dal Canto

Polchow

Alexander

Caliendo

Hentati

Kwon

Deng

(1994) Motor neuron degeneration in mice that express a human Cu, Zn superoxide dismutase mutation. Science 264:1772–1775

14.

Hall

Andrus

Oostveen

Fleck

Gurney

(1998) Relationship of oxygen radical-induced lipid peroxidative damage to disease onset and progression in a transgenic model of familial ALS. J Neurosci Res 53:66–77

15.

Huang

Panahian

Dalkara

Fishman

Moskowitz

(1994) Effects of cerebral ischemia in mice deficient in neuronal nitric oxide synthase. Science 265:1883–1885

16.

Jenner

Olanow

(1998) Understanding cell death in Parkinson's disease. Ann Neurol 44:S72–S84

17.

Keller

Germeyer

Begley

Mattson

(1997b) 17b-estradiol attenuates oxidative impairment of synaptic Na⁺/K⁺-ATPase activity, glucose transport, and glutamate transport induced by amyloid β-peptide and iron. J Neurosci Res 50:522–530

18.

Keller

Pang

Geddes

Begley

Germeyer

Waeg

Mattson

(1997a) Impairment of glucose and glutamate transport and induction of mitochondrial oxidative stress and dysfunction in synaptosomes by amyloid b-peptide: role of the lipid peroxidation product 4-hydroxynonenal. J Neurochem 69:273–284

19.

Kruman

Pedersen

Mattson

(1999) ALS-linked Cu/Zn-SOD mutation increases vulnerability of motor neurons to excitotoxicity by a mechanism involving increased oxidative stress and perturbed calcium homeostasis. Exp Neurol 160:28–39

20.

Liu

Althaus

Ellerbrock

Becker

Gurney

(1998) Enhanced oxygen radical production in a transgenic mouse model of familial amyotrophic lateral sclerosis. Ann Neurol 44:763–770

21.

Ludolph

Langen

Regard

Herzog

Kemper

Kuwert

Bottger

Feinendegen

(1992) Frontal lobe function in amyotrophic lateral sclerosis: a neuropsychologic and positron emission tomography study. Acta Neurol Scand 85:81–89

22.

Mark

Hensley

Butterfield

Mattson

(1995) Amyloid β-peptide impairs ion-motive ATPase activities: evidence for a role in loss of neuronal Ca²⁺ homeostasis and cell death. J Neurosci 15:6239–6249

23.

Mark

Lovell

Markesbery

Uchida

Mattson

(1997b) A role for 4-hydroxynonenal in disruption of ion homeostasis and neuronal death induced by amyloid β-peptide. J Neurochem 68:255–264

24.

Mark

Pang

Geddes

Uchida

Mattson

(1997a) Amyloid β-peptide impairs glucose uptake in hippocampal and cortical neurons: involvement of membrane lipid peroxidation. J Neurosci 17:1046–1054

25.

Mattson

(1997) Cellular actions of β-amyloid precursor protein, and its soluble and fibrillogenic peptide derivatives. Physiol Rev 77:1081–1132

26.

Mattson

Keller

Begley

(1998) Evidence for synaptic apoptosis. Exp Neurol 153:35–48

27.

Murai

Miyahara

Tanaka

Kaneko

Sako

Kameyama

(1983) Abnormalities of lipoprotein and carbohydrate metabolism in degenerative diseases of the nervous system—motor neuron disease and spinocerebellar degeneration. Tohoku J Exp Med 139:365–376

28.

Pedersen

Cashman

Mattson

(1999) The lipid peroxidation product 4-hydroxynonenal impairs glutamate and glucose transport and choline acetyltransferase activity in NSC-19 motor neuron cells. Exp Neurol 155:1–10

29.

Pedersen

Keller

Markesbery

Appel

Smith

Kasarskis

Mattson

(1998) Protein modification by the lipid peroxidation product 4-hydroxynonenal in the spinal cords of amyotrophic lateral sclerosis patients. Ann Neurol 44:819–824

30.

Reaume

Elliot

Hoffman

Kowall

Ferrante

Siwek

Wilcox

Flood

Beal

Brown

Scott

Snider

(1996) Motor neurons in Cu/Zn superoxide dismutasedeficient mice develop normally but exhibit enhanced cell death after axonal injury. Nat Genet 13:43–47

31.

Ripps

Huntley

Hof

Morrison

Gordon

(1995) Transgenic mice expressing an altered murine superoxide dismutase gene provide an animal model of amyotrophic lateral sclerosis. Proc Natl Acad Sci USA 92:689–693

32.

Rothstein

(1995) Excitotoxicity and neurodegeneration in amyotrophic lateral sclerosis. Clin Neurosci 3:348–359

33.

Rothstein

Van Kammen

Levey

Martin

Kuncl

(1995) Selective loss of glial glutamate transporter GLT-1 in amyotrophic lateral sclerosis. Ann Neurol 38:73–84

34.

Roy

Minotti

Dong

Figlewicz

Durham

(1998) Glutamate potentiates the toxicity of mutant Cu/Zn-superoxide dismutase in motor neurons by postsynaptic calcium-dependent mechanisms. J Neurosci 18:9673–9684

35.

Schapira

Taanman

Tabrizi

Seaton

Cleeter

Cooper

(1998) Mitochondria in the etiology and pathogenesis of Parkinson's disease. Ann Neurol 44(3 suppl 1):S89–S98

36.

Siklos

Engelhardt

Alexianu

Gurney

Siddique

Appel

(1998) Intracellular calcium parallels motoneuron degeneration in SOD-1 mutant mice. J Neuropathol Exp Neurol 57:571–587

37.

Trotti

Rolfs

Danbolt

Brown

Jr Hediger

(1999) SOD1 mutants linked to amyotrophic lateral sclerosis selectively inactivate a glial glutamate transporter. Nat Neurosci 2:427–433

38.

Wiedau-Pazos

Goto

Rabizadeh

Gralla

Roe

Lee

Valentine

Bredesen

(1996) Altered reactivity of superoxide dismutase in familial amyotrophic lateral sclerosis. Science 271:515–518

39.

Wong

Pardo

Borchelt

Lee

Copeland

Jenkins

Sisodia

Cleveland

Price

(1995) An adverse property of a familial ALS-linked SOD1 mutation causes motor neuron disease characterized by vacuolar degeneration of mitochondria. Neuron 14:1105–1116

40.

Yang

Chan

Chen

Carlson

Chen

Weinstein

Epstein

Kamii

(1994) Human copper-zinc superoxide dismutase transgenic mice are highly resistant to reperfusion injury after focal cerebral ischemia. Stroke 25:165–170

41.

Yim

Kang

Chock

Stadtman

Yim

(1997) A familial amyotrophic lateral sclerosis-associated A4V Cu, Zn-superoxide dismutase mutant has a lower Km for hydrogen peroxide. Correlation between clinical severity and the Km value. J Biol Chem 272:8861–8863

ALS-Linked Cu/Zn-SOD Mutation Impairs Cerebral Synaptic Glucose and Glutamate Transport and Exacerbates Ischemic Brain Injury

Abstract

Keywords

MATERIALS AND METHODS

Mice and transient focal cerebral ischemia protocol

Synaptosome preparation and experimental treatment

Measurement of glucose and glutamate transport and lipid peroxidation

RESULTS AND DISCUSSION

Footnotes

Acknowledgments

Abbreviations used

References