The expression of the glutamate re-uptake transporter excitatory amino acid transporter 1 (EAAT1) in the normal human CNS and in motor neurone disease: an immunohistochemical study.

A monoclonal antibody to excitatory amino acid transporter 1 (EAAT1) has been generated which robustly stains paraffin-embedded, formaldehyde-fixed as well as snap-frozen human post-mortem brain tissue. We have used this antibody to map the distribution of EAAT1 throughout normal human CNS tissue. In addition this antibody has been used to perform a semi-quantitative immunohistochemical analysis of the expression of EAAT1 in motor cortex and cervical cord tissue taken from motor neurone disease cases (n=17) and neurologically normal controls (n=12). By comparing the relative optical density measurements of identical regions of motor cortex and cervical spinal cord an increase in the expression levels of EAAT1 was observed in motor neurone disease tissue compared to the control tissue and in both motor cortex and cervical spinal cord (9-17% and 13-33% increases respectively). EAAT1 was observed to be the most abundant transporter in more "caudal" brain regions such as the diencephalon and brainstem and its expression in other regions was frequently more uniform than that of EAAT2. In the motor cortex, EAAT1 immunoreactivity was present in all grey matter laminae, with some staining of individual astrocytes in the white matter. In spinal cord, EAAT1 immunoreactivity was strongest in the substantia gelatinosa. In the ventral horn, motor neurones were surrounded with a dense rim of perisomatic EAAT1 immunoreactivity, and the neuropil showed diffuse staining. Additional studies using double-labelling immunocytochemistry demonstrated that astrocytic co-localisation of EAAT1 and EAAT2 may occasionally be seen, but was not widespread in the human CNS and that in general astrocytes were positive for either EAAT1 or EAAT2. These results demonstrate that the EAAT1 has a widespread abundance throughout all regions of the human CNS examined and that there exist discrete populations of astrocytes that are positive solely for either EAAT1 or EAAT2. Furthermore, there is evidence to suggest that altered EAAT1 expression in motor neurone disease follows a different pattern to the reported changes of EAAT2 expression in this condition, indicating that the role of glutamate transporters in the pathogenesis of motor neurone disease appears more complex than previously appreciated.

Glutamine synthetase activity and expression are not affected by the development of motor neuronopathy in the G93A SOD-1/ALS mouse.

The expression and activity of the enzyme glutamine synthetase (GS) were examined in the G93A/SOD-1 transgenic mouse model of progressive motor neuronopathy to investigate the mechanisms underlying degeneration of the motor neurones. Clinical signs appeared in G93A/SOD-1 mice at around 90 days, with severe spasticity and loss of self-righting reflex from 120 to 150 days of age. GS expression was examined using western blotting in primary astrocyte cultures derived from newborn (P1-2) G93A/SOD-1 mice and their non-transgenic littermates and in lower spinal cord from animals at 30, 60 and 90 days of age and disease end-stage (120-150 days). There were no differences in the levels of GS expression in the transgenic mice compared to the unaffected littermates at any of the disease stages examined. GS activity was measured spectrophotometrically in spinal cord extracts at these disease stages. There was a decrease in V(max) at 60 days compared to 30 days in both groups of mice (3.48+/-0.58 cf. 6.43+/-1.83 mmol/h/mg protein; non-transgenic littermates), with GS activity highest at end-stage (9.38+/-0.71 mmol/h/mg protein cf. 7.64+/-0.42 mmol/h/mg protein in littermates). Conversely, K(m) was transiently increased at 60 days (2.53+/-0.26 mM cf. 1.32+/-0.20 in littermates), remaining within the range of 30 day measurements from 90 days onwards. There were no differences in V(max) or K(m) values between the G93A/SOD-1 mice and their unaffected non-transgenic littermates at any of the disease stages examined. We conclude that there is no evidence that a change in glutamine synthetase activity or expression contributes to the progressive neurodegeneration observed in the G93A/SOD-1 mice.

Cultured glial cells are resistant to the effects of motor neurone disease-associated SOD1 mutations.

Free radical damage has been implicated in the pathophysiology of motor neurone disease (MND); mutations have been identified in the gene encoding Cu/Zn superoxide dismutase (SOD1). There is evidence that glial cell dysfunction may contribute to motor neurone injury, but the exact role of glial cells in MND has yet to be established. The aim of this study was to determine whether expression of mutant SOD1 affects the response of glia to oxidative stress. Stable C6 glioma cells expressing mutant SOD1 and cortical astrocyte cultures from G93A-SOD1 transgenic mice were exposed to: xanthine/xanthine oxidase; hydrogen peroxide; A23187 and 3-morpholinosydonimine. Cell viability was measured using the 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Neither C6 glioma cells nor cortical astrocytes expressing mutant SOD1 were more susceptible to any of the free radical generating systems compared to control cells. These results suggest that astrocytes are resistant to the toxic effects of mutant SOD1 widely reported for neuronal cells.

The RNA of the glutamate transporter EAAT2 is variably spliced in amyotrophic lateral sclerosis and normal individuals.

Impaired re-uptake of synaptic glutamate, and a reduced expression of the glutamate transporter EAAT2 have been found in the motor cortex of patients with amyotrophic lateral sclerosis (ALS). Two splice forms of the EAAT2 RNA resulting from retention of intronic sequences (EAAT2/Int) and deletion of one protein coding exon (EAAT2/C1) have been reported to account for the EAAT2 protein loss in ALS. In this study we investigated the presence of two known (EAAT2/C1; EAAT2/Int) and three novel (EAAT2/C2-4) EAAT2 RNA in motor cortex of 17 ALS cases and 11 controls. Reverse transcription and PCR were carried out to amplify the complementary DNA of the complete and variably spliced EAAT2 transcripts. Nested PCR was followed to generate amplicons specific for EAAT2/C1-4 and EAAT2/Int. EAAT2/Int was detected in 59% of ALS specimens as compared to 36% of controls showing a trend but no statistical significance of a more frequent expression in ALS (Type I error 24.6%). EAAT2/C1-4 were found to be equally expressed in ALS patients and controls. Our results indicate that the involvement of EAAT2 transcripts in ALS is unlikely to be primary, and more complex than previously recognized. Alterations of quantitative expression of distinct EAAT2 splice forms in ALS cannot be excluded from this study and remain to be investigated.

The role of astrocytes and noradrenaline in neuronal glucose metabolism.

In the classical model the energy requirements during neuronal activation are provided by the delivery of additional glucose directly into the extracellular compartment that results from the increase in local cerebral blood flow (rCBF). The present review proposes that astrocytes play a key role in the response to neuronal activation. Arginine for the synthesis of NO, which has a major role in the increase in rCBF, is released from astrocytes in response to stimulation of astrocytic glutamate receptors. The increased delivery of glucose by the blood stream enters astrocytes, where some of it is converted to glycogen. During neuronal activation there is a decrease in extracellular glucose owing to increased utilization followed by a delayed increase; this results from stimulation of astrocytic beta-adrenergic receptors, which leads to a breakdown of glycogen and the export of glucose.

The expression of the glial glutamate transporter protein EAAT2 in motor neuron disease: an immunohistochemical study.

Emerging evidence suggests that a disturbance of the glutamate neurotransmitter system may be a contributory factor to motor neuron injury in motor neuron disease. Previous autoradiographic and immunoblotting studies have suggested that there may be reduced expression of glutamate transporter proteins in pathologically affected areas of the CNS in motor neuron disease. This study further explores the possible alteration in expression of the excitatory amino acid transporter protein EAAT2 in MND, by examining the protein expression in situ, in frozen sections, using immunohistochemistry. The aim of the study was to compare the distribution and density of EAAT2 in the motor cortex and spinal cord of MND cases (n = 16) compared with neurologically normal controls (n = 12), matched for relevant parameters. A novel, previously characterized, monoclonal antibody to EAAT2 was employed. EAAT2 immunoreactivity in motor neuron disease and control cases was compared using relative optical density measurements generated by computerized image analysis. In the motor cortex, EAAT2 immunoreactivity was laminated comprising a superficial intense band (corresponding to layers 1 and 2); a paler middle band (layer 3 and part of 5) and a more intense deep layer (layers 5 and 6). In the spinal cord, the ventral horn showed strong immunoreactivity with dense perisomatic staining around motor neuron cell bodies, the substantia gelatinosa showed moderate diffuse staining and the intermediate spinal laminae showed weak staining. This general pattern of immunoreactivity was preserved in the motor neuron disease cases. However, in the motor neuron disease cases compared with controls, the optical density values for EAAT2 immunoreactivity were significantly reduced in all grey matter regions of the lumbar spinal cord (P < 0.001) and were increased in the middle laminae of the motor cortex (P < 0.05). This study indicates that glutamate transporter pathology in motor neuron disease may be a more complex phenomenon than previously recognized.

Extracellular glucose turnover in the striatum of unanaesthetized rats measured by quantitative microdialysis.

1. Steady-state and time-resolved quantitative microdialysis was used to measure dialysate concentration, extracellular concentration and the in vivo recovery of glucose in rat striatum. 2. The extracellular concentration of glucose, determined by the zero net flux method of Lönnroth, was 350 +/- 20 microM and the in vivo recovery was 39 +/- 2%. 3. Veratridine caused a steep decrease in dialysate glucose after an initial delay of 7.5 min. When steady-state glucose levels had been reached in the presence of veratridine the extracellular concentration was reduced to zero, but there was no significant change in in vivo recovery. 4. Measurement of the dynamic changes during the administration of veratridine showed an immediate decrease in extracellular glucose concentration and a steep rise in in vivo recovery, which accounted for the delay in the delay in the decrease in dialysate glucose. When extracellular concentration reached zero, in vivo recovery returned to control levels.

Expression of the glial glutamate transporter EAAT2 in the human CNS: an immunohistochemical study.

Glutamate transporters play an essential role in terminating the excitatory glutamatergic signal at post-synaptic receptors and in protecting neurones from excitotoxic effects, as well as replenishing the neurotransmitter supply at glutamatergic synapses. The distribution and density of glutamate transporters may be important determinants of vulnerability to glutamate-mediated injury. There is emerging evidence that glutamate transporter dysfunction may be present in motor neurone disease (MND). In this study, a monoclonal antibody, suitable for immunohistochemistry (IHC) in human post-mortem tissue, was produced to the human astrocytic glutamate transporter EAAT2 (excitatory amino acid transporter 2). Western blotting of homogenates of human cortical tissue with the EAAT2 antibody produced a discrete band at 66 kDa. Detailed IHC analysis of the expression of the EAAT2 protein in the human CNS was undertaken. EAAT2 was exclusively localised to astrocytes, with preferential expression in the caudate nucleus, nucleus basalis of Meynert, spinal ventral horn, cerebral cortex and hippocampus, but with lower levels of expression throughout many other CNS regions. Motor neurone groups vulnerable to neurodegeneration in MND appeared distinctive in being surrounded by extensive, coarse, strongly immunoreactive perisomatic glial profiles. Motor neurone groups which tend to be spared in MND, such as those present in the oculomotor nucleus, showed a lower expression of EAAT2, with fewer perisomatic profiles. The EAAT2 antibody will provide a useful tool for increasing our understanding of the role of EAAT2 in excitatory neurotransmission in health and disease states.

The mechanisms controlling physiologically stimulated changes in rat brain glucose and lactate: a microdialysis study.

1. This study is concerned with the supply of metabolic substrates for neuronal metabolism. Experiments were carried out to investigate whether mechanisms demonstrated in cultured astrocytes also occurred in vivo; these were cAMP-mediated breakdown of glycogen and glutamate uptake-stimulated release of lactate. 2. In vivo microdialysis was used in freely moving rats. Lactate and glucose in the dialysate were assayed using enzyme-based on-line assays. Drugs were given locally through the dialysis probe. Regional cerebral blood flow was measured using the hydrogen clearance method. 3. There was an increase in dialysate glucose in response to the beta-adrenoceptor agonist isoprenaline and to 8-bromo-cAMP, an analogue of cAMP, the second messenger of beta-adrenoceptor stimulation. The effect of isoprenaline was blocked by the antagonist propranolol. Isoprenaline had no effect on dialysate lactate, which was increased by the glutamate uptake blocker beta-D,L-threohydroxyaspartate (THA). 4. Physiological stimulation of neuronal activity produced an increase in both lactate and glucose. The increase in lactate was depressed in the presence of THA but was unaffected by propranolol. The increase in glucose was blocked by propranolol. Regional cerebral blood flow was increased by physiological stimulation but was unaffected by propranolol. 5. These results demonstrate that physiologically stimulated increases in glucose and lactate in the brain are mediated by different mechanisms.