Brain, Vol. 125, No. 8, 1908-1922,
August 2002
© 2002 Guarantors of Brain
Impaired glutamate transport and glutamate–glutamine cycling: downstream effects of the Huntington mutation
1 Department of Neurology, Universitätsklinik Freiburg, 2 Department of Neurology, Universitätsklinik Ulm, Germany and 3 Division of Medical and Molecular Genetics, Guy’s Hospital, London, UK
Correspondence to: Peter F. Behrens, MD, Department of Neurology, Universitätsklinik, Breisacherstrasse 64, 79106 Freiburg, Germany E-mail: pfbehrens{at}web.de
Received July 23, 2001. Revised February 14, 2002. Accepted February 25, 2002.
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The pathogenesis of Huntington’s disease is still not completely understood. Several lines of evidence from toxic/non-transgenic animal models of Huntington’s disease suggest that excitotoxic mechanisms may contribute to the pathological phenotype. Evidence from transgenic animal models of Huntington’s disease, however, is sparse. To explore potential alterations in brain glutamate handling we studied transgenic mice expressing an N-terminal fragment of mutant huntingtin (R6/2). Intracerebral microdialysis in freely moving mice showed similar extracellular glutamate levels in R6/2 and littermate controls. However, partial inhibition of glutamate transport by L-trans-pyrrolidine-2,4-dicarboxylate (4 mM) disclosed an age-dependent increase in extracellular glutamate levels in R6/2 mice compared with controls, consistent with a reduction of functional glutamate transport capacity. Biochemical studies demonstrated an age-dependent downregulation of the glial glutamate transporter GLT-1 mRNA and protein, resulting in a progressive reduction of transporter function. Glutamate transporters other than GLT-1 were unchanged. In addition, increased extracellular glutamine levels and alterations to glutamine synthetase immunoreactivity suggested a perturbation of the glutamate–glutamine cycle. These findings demonstrate that the Huntington’s disease mutation results in a progressively deranged glutamate handling in the brain, beginning before the onset of symptoms in mice. They also provide evidence for a contribution of excitotoxicity to the pathophysiology of Huntington’s disease, and thus Huntington’s disease may be added to the growing list of neurodegenerative disorders associated with compromised glutamate transport capacity.
Keywords: glutamate transport; glutamate transporter; Huntington’s disease; mutant huntingtin; transgenic mouse
Abbreviations: DHK= dihydrokainate; EAAC1 = excitatory amino acid carrier 1; GAPDH = glyceraldehyde-3-phosphate dehydrogenase; GLAST = L-glutamate/L-aspartate transporter; GLT = glutamate transporter; GS = glutamine synthetase; HD gene = Huntington’s disease gene; mGluR = metabotropic glutamate receptor; PAG = phosphate-activated glutaminase; PDC = L-trans-2,4-pyrrolidine dicarboxylate
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Introduction |
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Huntington’s disease is an autosomal dominant neurodegenerative disorder, clinically characterized by personality changes, a movement disorder and a decline in cognitive function, typically with onset in midlife (Harper, 1991
). Huntington’s disease is caused by an expansion of a CAG trinucleotide repeat within exon 1 of the Huntington’s disease gene (HD gene) on chromosome 4 (Huntington’s Disease Collaborative Research Group, 1993). The mutant HD gene encodes an extended polyglutamine (poly-Q) stretch in the N-terminal domain of a protein of unknown function called huntingtin. Pathologically, the hallmark of Huntington’s disease is an accumulation of poly-Q-expanded huntingtin fragments in the form of nuclear and cytoplasmatic inclusions, a generalized brain atrophy and a selective neuronal loss (Vonsattel and DiFiglia, 1998). Neuronal loss accompanied by fibrillary gliosis is most obvious in the striatum, where striatal projection neurones are preferentially lost (Albin et al., 1992). To date it is still unclear how the Huntington’s disease mutation results in neuronal dysfunction and, finally, selective neuronal loss.
Based on the observation that in experimental animals, intrastriatal injections of N-methyl-D-aspartate (NMDA) receptor agonists such as quinolinic acid mimicked many of the pathological and neurochemical features of Huntington’s disease (Beal et al., 1991), excitotoxic mechanisms were hypothesized to play a role in its pathogenesis (Albin and Greenamyre, 1992; Beal, 1992). In addition, differences in the expression pattern of glutamate receptors were proposed to contribute to the selective vulnerability of striatal projection neurones and the relative resistance of striatal interneurones (Landwehrmeyer et al., 1995). Mechanisms directly linking the Huntington’s disease mutation with glutamatergic dysfunction have not been identified to date, however.
Transgenic mouse models of Huntington’s disease allow us to study its pathogenesis. Studies using the mouse line R6/2, expressing an N-terminally truncated huntingtin containing 141–157 poly-Qs under the control of the human Huntington’s disease promoter (Mangiarini et al., 1996), demonstrated that a nuclear accumulation of mutant huntingtin altered the expression of a distinct set of genes (Cha et al., 1998; Luthi-Carter et al., 2000). Among others, expression levels of a metabotropic glutamate receptor, mGluR2, were markedly decreased (Cha et al., 1998). This receptor is known to be presynaptically localized on corticostriatal terminals (Testa et al., 1998) and is thought to regulate glutamate release (Cartmell and Schoepp, 2000). Reduced expression of this receptor might therefore result in impaired feedback control with increased synaptically released glutamate. Since even slight increases of extracellular glutamate concentrations may lead to neuronal degeneration (Beal, 1992) we explored the levels of extracellular glutamate in the course of the developing disease.
We performed a microdialysis study in the striatum and cortex of freely moving R6/2 mice of three different ages, determining basal levels of glutamate and exploring the effect of the selective competitive uptake inhibitor L-trans-2,4-pyrrolidine dicarboxylate (PDC). This study has demonstrated that PDC induced larger increases in extracellular glutamate levels in transgenic animals than in controls, suggesting either a reduced capacity for glutamate uptake or an excessive release. We therefore examined the glutamate uptake system in R6/2 mice and discovered a progressive downregulation of the expression of the glial glutamate transporter GLT-1.
In addition, we observed elevated extracellular glutamine levels, suggesting an alteration in the glutamate–glutamine cycle (Martinez-Hernandez et al., 1977; Hertz, 1979; Daikhin and Yudkoff, 2000). We therefore studied the two key enzymes of this cycle: glutamine synthetase (GS), which converts glutamate to glutamine within glial cells, and phosphate-activated glutaminase (PAG), which hydrolyses glutamine to glutamate within the mitochondria of synaptic terminals.
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Animals
The experimental protocol was in accordance with German laws for animal protection and was approved by the local Review Board for Animal Research (Regierungspräsidium Freiburg, Germany).
Female and male R6/2 mice, transgenic for a mutant exon 1 of the HD gene, and their normal littermate controls (wild type) were obtained from G. Bates (Division of Medical and Molecular Genetics, Guy’s and St Thomas’ Hospital, London, UK). All mice were genotyped using tail biopsies and a polymerase chain reaction (PCR) assay as described previously (Mangiarini et al., 1996). Animals were housed in a 12-h light–dark cycle environment and given food pellets and water ad libitum. Body weights were in the range of 18–22 g.
Microdialysis procedure
Microdialysis studies were performed in 72 R6/2 and control mice of 6, 9 and 12 weeks of age. The microdialysis system used in our studies was manufactured by CMA/Microdialysis (CMA, Solna, Sweden). Straight microdialysis probes, with an outer shaft diameter of 0.18 mm, equipped with a Cuprophane membrane (length 1 mm, molecular cut-off 
6 kDa; MAB 4, Metalant, Sweden) were used.
All animals were operated on under anaesthesia with isoflurane 1.5–3% in oxygen. One guide cannula was implanted in each mouse as follows. In the first group it was implanted in the right striatum according to a stereotaxic mouse atlas (Franklin and Paxinos, 1997) at the following coordinates: at the level of the bregma, 2 mm lateral to the midline and 2 mm below the dural surface. In the second animal group it was implanted vertically in the right sensorimotor cortex at the level of the bregma, 2 mm lateral to the midline and 0.2 mm below the dural surface. Dialysis was performed the next day when the mice had recovered from the implantation procedure.
The probes were perfused continuously with a modified degassed Ringer’s solution containing Na+ 127 mmol/l, K+ 2.5 mmol/l, Ca2+ 1.3 mmol/l, Mg2+ 0.9 mmol/l (pH 7.4) at a flow rate of 1.5 ml/min. After a 60 min equilibration period, consecutive 20 ml fractions of perfusate were collected. Preliminary studies demonstrated that extracellular amino acid concentrations had stabilized after 60 min. Samples were either analysed immediately or stored at –80°C. After the first three fractions with perfusion of modified Ringer’s solution (basal levels of amino acids), PDC (Tocris, Langford, Bristol, UK) 4 mM (60 animals) or dihydrokainate (DHK; Tocris, Langford, Bristol, UK) 10 mM (12 animals), dissolved in modified Ringer’s solution, was perfused for a further three fractions. Finally, another three fractions were collected with solely modified Ringer’s perfusion medium.
Throughout all of the experiments the body temperature of the animals was maintained at 38.5°C by means of a thermostatically controlled heating pad.
In vitro recovery was determined before the experiments, mainly to control the experimental error due to the variation in probe construction. Probes showing >15% deviation from the mean recovery value for glutamate were discarded.
Amino acid analysis by high performance liquid chromatography
Amino acid analysis was performed by high performance liquid chromatography (HPLC) with gradient elution and pre-column derivatization using a modification of the method described by Godel and Graser (Graser et al., 1985; Behrens et al., 2000). Amino acids and HPLC solvents were obtained from either Sigma (Deisenhofen, Germany) or Merck (Darmstadt, Germany). As 
-aminobutyric acid (GABA) could not be reliably measured in all samples, it was not included in the analysis.
All amino acid concentrations are given as dialysate concentrations and were not corrected for recovery.
After completion of the microdialysis experiments, the animals were deeply anaesthetized with isoflurane 5% in oxygen and decapitated. Brains were removed, covered with embedding medium, frozen at –30 to –40°C in isopentane then kept frozen at –80°C until sectioning. To assess the accuracy of probe insertion, sections (12 µm) were cut coronally in a cryostat and stained with haematoxylin–eosin. Probe placement and histological damage were determined by microscopic inspection. Only animals with correct probe placement were included in the analysis (Fig. 1A and B).
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Construction of a cRNA probe for the glutamate transporter GLT-1
A cRNA probe recognizing a coding sequence unique to GLT-1 but shared by all known splice variants of GLT-1 was used in this study. The probe was derived from a template generated by PCR amplification of ‘mouse Marathon Ready cDNA’ (Clontech, Heidelberg, Germany) using the following primers containing T7 and SP6 RNA polymerase promoter sequences, respectively. P1: forward primer 5'-CTG TAA TAC GAC TCA CTA TAG GGG GCC TCA TCA TTC ACG GG-3'; reverse primer 5'-GGG ATT TAG GTG ACA CTA TAG AAA CTG ATA TCC TCC GTT GG-3'). PCR products were purified using a PCR purification kit (Qiagen, Hilden, Germany). RNA probes were synthesized by in vitro transcription as described previously (Kerner et al., 1998
). Incorporation of the 35S-CTP was measured with a scintillation counter. The probe was used within 1 day.
In situ hybridization histochemistry
In situ hybridization histochemistry studies were performed in five R6/2 and five control mice, all of which were 12 weeks of age. Frozen coronal sections (20 µm thick) were cut on a cryostat (Leica, Bensheim, Germany), thaw-mounted on poly-L-lysine-coated slides and stored at –80°C until used. The in situ hybridization histochemistry of striatal sections was performed as described in Landwehrmeyer et al. (1995). Briefly, sections were fixed in 4% PFA–PBS (paraformaldehyde–phosphate-buffered saline), washed in PBS, acetylated, dehydrated, delipidated in chloroform, partially rehydrated and air dried. Sections were hybridized for 4 h at 50°C with the 35S-labelled probe. After hybridization, sections were washed, treated with RNase A and partially dehydrated. Slides were exposed to HyperfilmTM ß-max (Amersham Pharmacia Biosciences Europe, Freiburg, Germany) for 5 days. Film autoradiograms are read using all wavelengths within the visible spectrum. Northern light ensures uniform luminescence throughout a reading session but does not select certain wavelengths.
Antibodies
Monoclonal antibodies to GS (Chemicon, Hofheim, Germany) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Chemicon, Hofheim, Germany) were used. In addition, polyclonal antibodies to GLT-1 (affinity purified; kindly provided by J. D. Rothstein, Johns Hopkins University, Baltimore, MD, USA), PAG (kindly provided by N. P. Curthoys, Colorado State University, Fort Collins, CO, USA), EAAC1 (Alpha-diagnostics, San Antonio, TX, USA) and GLAST (Chemicon, Hofheim, Germany) were employed. Specificity was assessed using western blots and/or immunocytochemistry on cells.
Immunohistochemistry
Immunohistochemistry studies were performed in two R6/2 and two control mice, all of which were 12 weeks of age. Mice were transcardially perfused with 4% PFA in PBS. Brains were removed, post-fixed in 70% ethanol at 4°C and paraffin embedded. Coronal, sagittal and horizontal sections were cut at a thickness of 5 µm. Sections were stained by standard immunohistochemical methods. Briefly, following deparaffination, slides were rehydrated in ethanol and boiled in citrate buffer (Oliver et al., 1997). Endogenous peroxidase was blocked with methanol/H2O2, then after washing the slides were treated with PBS/0.3% Triton X-100/5% normal goat serum (primary polyclonal rabbit antibody), or 5% normal horse serum (primary monoclonal mouse antibody). After blocking, sections were incubated with the primary antibodies overnight at 4°C (GLT-1 1 : 200; GLAST 1 : 200; EAAC1 1 : 200; GS 1 : 250; PAG 1 : 100) in PBS containing 3% horse serum. Biotinylated secondary antibodies (1 : 200) were visualized using the avidin–biotin–peroxidase complex method (Vectastain Elite Kit; Vector Laboratories, Burlingame, CA, USA) using DAB (diaminobenzidine acid) as chromogen. Some sections were lightly counterstained with haematoxylin. Sections were dehydrated and mounted on coverslips.
Quantitative immunoblotting
Quantitative immunoblotting studies were performed in five R6/2 and five control mice, all of which were 12 weeks of age. The striata and the overlying sensorimotor cortex were rapidly dissected out at 4°C, and homogenized in 25 mM Tris–HCl containing 1% SDS and a protease inhibitor cocktail (aprotinin, leupeptin, phenylmethylsulfonylfluoride; Sigma). After sonication for 30 s, the homogenates were centrifuged for 10 min at 10 000 g and the particulated material resuspended in the loading buffer. The protein concentrations were determined by the Bradford assay (Bio-Rad, Munich, Germany). Equal amounts of protein (20 µg/lane) were loaded onto 12.5% SDS polyacrylamide gels, electrophoresed and blotted onto nitrocellulose membranes (0.45 µm pore size; Schleicher und Schuell, Dassel, Germany). Blots were incubated with the following antibody concentrations: GLT-1 1 : 500, EAAC 1 : 500, GLAST 1 : 400, PAG 1 : 400, GS 1 : 1000, GAPDH 1 : 8000. After incubation with horseradish peroxidase-labelled secondary antibodies (Sigma-Aldrich, Taufkirchen, Germany), the signal was visualized on film with enhanced chemoluminescence (ECL; Amersham Pharmacia Biosciences Europe, Freiburg, Germany). Blots were done in duplicate.
The resulting bands were digitized (MCID; Imaging Research, St Catherine’s, Ontario, Canada) and the band intensities were calculated as the product of area of the band x relative optical density – background. The ratio of band intensities of R6/2 to controls was calculated. GAPDH was used as an endogenous control of protein loading onto the gel.
Glutamate uptake assay
Uptake studies were performed in five R6/2 and five control mice, each of which were 12 weeks of age. Crude synaptosomes were prepared from the mice forebrains according to modified protocols from Robinson et al. (1993) and Ganel and Crosson (1998). In short, the brains were dissected and homogenized in sucrose 0.32 M, Tris buffer pH 7.4. After washing, the synaptosomes were incubated for 5 min at 30°C with 3H-labelled glutamate (specific activity 42.0 Ci/mmol glutamate; Amersham) at a total glutamate concentration of 10 µM. Radioactivity was determined without and after preincubation (15 min) with 1 mM DHK as specific inhibitor of GLT-1. The assays were harvested and washed on filters using Millipore collectors, membranes were solubilized, and radioactivity determined by scintillation counting. Each assay was performed in triplicate and results expressed in counts per minute per mg/ml of protein. DHK-dependent uptake was calculated as the difference between glutamate uptake with and without preincubation with DHK.
Statistical analysis
All values are expressed as means ± SD. For the microdialysis experiments and the in situ hybridization study, statistical analysis was performed using a three-way repeated measures ANOVA (analysis of variance) for comparisons between groups and single cells. The effect of PDC on dialysate glutamate was analysed using the difference between PDC-induced glutamate levels and basal glutamate.
Analysis of immunoblots and of the glutamate uptake assay was performed using Student’s t-test. A P value <0.05 was considered significant.
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Microdialysis: glutamate
Basal glutamate levels were similar in R6/2 and control mice at all ages examined (Table 1; Fig. 2). There were no regional differences in basal glutamate levels. When PDC (4 mM) was perfused through the dialysis probe, extracellular concentrations of glutamate rose steeply, stayed at a high level as long as PDC was perfused and then declined rapidly towards basal levels after discontinuation of PDC perfusion (Fig. 2A). Increase of glutamate in the dialysate was dependent on PDC concentration in both wild-type and R6/2 mice (Fig. 2B). In 12-week-old R6/2 mice, extracellular concentrations of glutamate increased to significantly higher levels than in wild-type animals in both striatum (1.9-fold) and cortex (1.6-fold) (Fig. 3). The enhanced increase in extracellular glutamate in R6/2 mice was age dependent: 6-week-old R6/2 and control mice showed increases of dialysate glutamate; however, at 9 and 12 weeks transgenic animals demonstrated a significantly enhanced response to PDC (Fig. 3). Response to PDC in control animals was independent of age in all three groups studied. Reverse microdialysis with PDC (4 mM) did not induce behavioural changes in any of the animals. In particular, no tonic/clonic epileptic seizures, barrel rotations, chewing or tonic movements of the forelimbs were observed. Histological analysis did not disclose obvious signs of neuronal damage other than the lesion caused by the needle track (data not shown).
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In addition, in 12 animals, each of which were 12 weeks of age, 10 mM DHK, a non-transportable inhibitor of glutamate uptake, was used to increase extracellular glutamate levels by reverse dialysis through the microdialysis probe. In these animals glutamate increase was more enhanced in wild-type than R6/2 mice; however, the absolute increase in extracellular glutamate levels was lower compared with PDC (Table 2). Unfortunately, DHK induced major behavioural abnormalities and seizures in the already seizure-prone animals, therefore a larger series of animals could not be examined and statistics were not performed for these experiments.
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Microdialysis: glutamine, taurine, alanine, glycine and serine
At 6 weeks, basal extracellular glutamine in the dialysate was not significantly different in R6/2 and control animals. However, the difference between R6/2 and controls increased significantly with age in both striatum and cortex (Fig. 4). The mean glutamine dialysate levels of cortex and striatum in R6/2 mice at 12 weeks of age were 1.8-fold above the levels of the wild-type control animals.
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Taurine showed an even greater rise in R6/2 mice, increasing progressively in both striatum and cortex, whereas taurine levels in control animals showed a modest decline with age (Fig. 5). At 12 weeks of age the mean taurine dialysate concentration in cortex and striatum was increased 4.9-fold in the R6/2 mice compared with the littermate controls. Glycine, serine and alanine dialysate levels were similar in R6/2 and wild type at all time points (data not shown).
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Glutamate transporter in situ hybridization histochemistry, immunoblotting, immunohistochemistry and uptake assay
In control animals and R6/2 mice, GLT-1 in situ hybridization histochemistry (ISHH) signals were evenly distributed throughout the striatum and displayed a layered pattern in cerebral cortex with somewhat more intense signals in the upper cortical layers (Fig. 6). In R6/2 mice, ISHH signals obtained with the GLT-1 probe were already significantly diminished in 6-week-old animals to 50 and 52% of control in striatum and cortex, respectively (Fig. 7). GLT-1 hybridization signals decreased further as the transgenic animals aged: at 12 weeks, GLT-1 signals were reduced to 32% (striatum) and 24% (cortex) of the signals obtained in littermates of the same age. The apparent loss of mRNA expression for GLT-1 in aging R6/2 animals was accompanied by a corresponding decrease of GLT-1 protein levels. In 12-week-old animals, quantitative immunoblotting showed significantly reduced GLT-1 protein levels: in striatum to 59% and cortex to 41% compared with controls (Fig. 8). Immunohistochemically, the distribution of GLT-1 immunoreactivity was similar in controls and R/2, demonstrating a patchy pattern throughout the cortex and striatum with a homogenous reduction of GLT-1 immunoreactivity in R6/2 (Fig. 9). We found no evidence for morphological changes or a different subcellular distribution of immunoreactivity in astrocytes of R6/2 expressing GLT-1. The overall number of GLT-1-expressing cells seemed unchanged, therefore the overall decrease of GLT-1 protein seemed to be due to a decrease in protein content per cell.
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GLAST and EAAC1 protein levels in contrast were similar in striatum and cortex of R6/2 and wild-type mice at 12 weeks of age (Fig. 8). GLAST immunostaining in cortex and striatum appeared similar in R6/2 and control mice. EAAC1 immunostaining was displayed intensely in soma and neuropil of cortical neurones. In R6/2 cells, however, somata appeared smaller and more irregular (Fig. 9).
The transporter assay showed a DHK (1 mM)-dependent glutamate uptake of 30.7 ± 8.1 µM glutamate/mg protein in wild type and 23.4 ± 5.3 µM glutamate/mg protein in R6/2 mice. This corresponds to a 24% decrease of uptake in the transgenic mice (P = 0.02).
PAG, GSA immunoblotting and immunohistochemistry
In the 12-week-old mice, immunohistochemistry using an antibody to PAG showed a homogenous staining pattern in the grey matter of R6/2 and wild-type animals, indicating a preferential labelling of neuropil. There was no obvious difference in the distribution or the intensity of the immunohistochemical signals between controls and R6/2 mice. Immunoblotting showed similar levels of protein in the transgenic and control animals in striatum and cortex (Fig. 8).
Similarly, immunoblotting demonstrated that regional protein levels of GS in both striatum and cortex were equal in 12-week-old R6/2 mice and control littermates (Fig. 8). Immunohistochemistry demonstrated, however, that the overall density of GS-immunoreactive cells was decreased while a subset of glial cells displayed enlarged processes with intense immunoreactivity (Fig. 9).
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Discussion |
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This study demonstrates that in a transgenic mouse model of Huntington’s disease, glutamate transport is impaired by a downregulation of the glial glutamate transporter GLT-1 at both the mRNA and protein levels, resulting in a progressive reduction of function. Microdialysis in freely moving mice showed that despite an impaired glutamate transport, extracellular glutamate levels were still within the normal range, suggesting that in R6/2 mice the downregulation of GLT-1 is functionally compensated. Partial inhibition of glutamate transport by PDC, however, disclosed an age-dependent increase in extracellular glutamate levels in R6/2 mice compared with controls, consistent with a reduction of functional glutamate transport capacity. In addition, the notion that glial function is altered in R6/2 mice is supported by increased extracellular glutamine levels, indicating a perturbation of the glutamate–glutamine cycle, and by increased taurine levels suggesting a disordered taurine metabolism. Taken together these data indicate that glutamate handling is perturbed in R6/2 brains.
Extracellular glutamate levels in R6/2 mice
The rise of dialysate glutamate concentrations evoked by PDC was substantially larger in R6/2 mice than in controls. A similarly enhanced rise of extracellular glutamate levels following stimulation with K+ by reverse microdialysis was shown by Brundin’s group (Haraldsson et al., 2000). The increased rise of extracellular glutamate concentration on application of PDC may indicate either a larger than normal release of glutamate or an impaired clearance of glutamate from the extracellular compartment. PDC is a transportable glutamate analogue that produces a dose-dependent elevation of extracellular glutamate levels without interfering with glutamate receptor binding (Massieu et al., 1995; Zuiderwijk et al., 1996; Massieu and Tapia, 1997; Segovia et al., 1997; Bridges et al., 1999). The increase in extracellular glutamate induced by infusion of 4 µM PDC in our control mice was similar to previously reported values in freely moving rats using the same dosage of PDC (Massieu et al., 1995). The mechanism of action of PDC is thought to involve at least two components: first, PDC acts as a selective inhibitor of high-affinity, sodium-dependent glutamate transport (without displaying specificity for any of the known five glutamate transporter molecules). Secondly, PDC releases glutamate from a cytosolic pool by heteroexchange. This second effect has been suggested to predominate in an acute ischaemia model in cell culture (Volterra et al., 1996) and in chronic neurodegeneration induced by PDC (Lievens et al., 1997). Preliminary evidence suggests that this second mechanism of action of PDC, heteroexchange, is also critical for our finding in R6/2 mice. Thus inhibition of glutamate uptake by the non-transportable glutamate transporter inhibitor DHK, which induces no heteroexchange, does result in even higher glutamate increases in controls than in R6/2 mice. However, the rise in extracellular glutamate induced by DHK was smaller than the one induced by PDC, raising the possibility that the apparent difference between DHK and PDC may reflect a less pronounced stress on the glutamate clearance mechanisms. A more thorough study using other non-transportable glutamate transporter inhibitors [DL-threo-ß-benzoylaspartate (DL-TBOA)] (Bridges et al., 1999) could not be done because these inhibitors interfered with the chromatographic determination of amino acids.
An increased glutamate release from either the glial or the neuronal compartment could be masked by efficient glutamate reuptake, but may become measurable following pharmacological antagonization of glutamate reuptake. An excessive rise of extracellular glutamate would be favoured by increased glutamate contents in R6/2 astrocytes. The intracellular glutamate concentration of R6/2 astrocytes is unknown. However, circumstantial evidence argues against a raised intracellular glutamate content of R6/2 astrocytes. Glutamate concentrations measured in tissue samples of different brain regions including the striatum in R6/2 were found to be similar to controls (Reynolds et al., 1999), or were even decreased (–24%) (Jenkins et al., 2000) like in Huntington’s disease striatum (Reynolds and Pearson, 1987). Glutamate contents of brain homogenates are difficult to interpret, however, since the contribution of glial glutamate to the overall tissue glutamate content may be too small to detect a functionally important redistribution. In addition, raised extracellular glutamine levels argue that the conversion of glial glutamate into glutamine is largely preserved in R6/2 brain.
Alternatively, there may be an excessive release of glutamate from the neuronal compartment in R6/2 mice. The PDC-induced glutamate increase may stimulate postsynaptic glutamate receptors which, via positive feedback, leads to further presynaptic glutamate release (Rawls and McGinty, 1997). This positive feedback loop may be sensitized in R6/2 mice, for example by enhanced sensitivity to postsynaptic glutamate receptor stimulation (Levine et al., 1999) or by diminished presynaptic autoinhibition secondary to a downregulation of mGluR expression (Cha et al., 1998). Since extracellular concentrations as determined by microdialysis do not necessarily reflect synaptically released glutamate, further electrophysiological studies measuring miniature potentials are required to clarify this issue. Finally, there may be an excessive non-synaptic release of neuritic glutamate as suggested by Li et al. (2000) as a consequence of an interference of cytoplasmic deposits of mutant huntingtin with the function of synaptic vesicles (Bates and Eberwine, 2000; Li et al., 2000).
Aside from an enhanced release, the differential effect of PDC in control and R6/2 mice may be explained by an impaired clearance of glutamate from the extracellular compartment. Indeed, an altered function of the glutamate transporter GLT-1 was demonstrated in transgenic animals and is discussed in more detail in the following paragraph. Based on the present data it is difficult to decide whether enhanced release or impaired clearance accounts for the excessive increase in extracellular glutamate following application of PDC in R6/2 mice. Both mechanisms are not mutually exclusive and may act in combination. We favour the view, however, that impairment of glutamate uptake is the predominant effect in R6/2 mice.
Selective downregulation of GLT-1 in R6/2 brains
In R6/2 mice we observed a decreasing GLT-1 mRNA expression, which was accompanied by a reduction in GLT-1 protein expression and a diminished DHK-sensitive glutamate uptake. An impairment of GLT-1 expression was apparent early on: in brains of 6-week-old animals, GLT-1 expression was reduced by 50%. The downregulation of GLT-1 mRNA and protein expression was uniform in R6/2 brains, indicating an effect on all glial cells rather than a loss of expression in a subset of glial cells or loss of a population of glial cells. These findings correspond to similar observations in Huntington’s disease brains, where decreased transcript levels of GLT-1 were found in the striatum (Arzberger et al., 1997) as well as reduced high-affinity uptake sites for glutamate (Cross et al., 1986). The downregulation of glutamate transporters appears to be limited to GLT-1, as protein levels of GLAST or EAAC1 were unchanged. GLT-1 is thought to account for the uptake of up to 90% of glutamate in the extracellular space (for reviews see Seal and Amara, 1999; Anderson and Swanson, 2000). Reduction of GLT-1 expression resulted in neuronal damage (Rothstein et al., 1996) and was associated with neuronal loss in amyotrophic lateral sclerosis (Rothstein et al., 1992; Trotti et al., 1999), Alzheimer’s disease (Masliah et al., 1996), hippocampal sclerosis (Mathern et al., 1999) and global transient cerebral ischaemia (Torp et al., 1995). In addition, transgenic mice deficient in GLT-1 show lethal spontaneous seizures, which also occurs frequently in R6/2 mice (Tanaka et al., 1997).
There are several ways in which GLT-1 expression could be downregulated in R6/2 mice. First, GLT-1 expression could be impaired by a primary glial pathology directly induced by the Huntington’s disease mutation. In Huntington’s disease, poly-Q expanded fragments of huntingtin aberrantly localize to and accumulate in the nuclear compartment and have been shown to alter the expression of a selective set of genes (for review see Cha, 2000). For instance, a selective downregulation of the neuronal glutamate transporter EAAT4 was found in Purkinje cells of mice transgenic for SCA1 (Lin et al., 2000). Nuclear poly-Q inclusions are detectable in R6/2 mice, not only in neurones but also in glial cells (Davies et al., 1999), raising the possibility that the transcriptional dysregulation demonstrated in neurones might occur in glia as well. Aside from a potential direct interaction of the mutant huntingtin with GLT-1 expression, a downregulation may involve a disordered cellular signalling: in cell culture cAMP analogues, modulated GLT-1 protein expression (Swanson et al., 1997) and signalling pathways involving cAMP have been shown to be impaired in R6/2 (Luthi-Carter et al., 2000). Secondly, GLT-1 expression could be reduced as a consequence of a primary neuronal impairment. Several studies demonstrated that GLT-1 expression is modified by neurones and that GLT-1 is undetectable in primary astrocyte cultures devoid of neurones (Zelenaia et al., 2000). Central axotomy downregulated GLT-1 expression in disconnected areas in several models (Ginsberg et al., 1995, 1996; Levy et al., 1995; Rao et al., 1998). Conflicting data from other studies probably reflect the fact that depending on the mechanisms by which neurones were injured, both a down- and an upregulation of GLT-1 expression could be observed (Lievens et al., 1997). Overall these data suggest that neurones release signalling factors regulating glial GLT-1 expression. Recent data suggest that pituitary adenylate cyclase-activating polypeptide, a neurone-derived peptide, plays a major role in regulating GLT-1 expression (Figiel and Engele, 2000). In addition, there is evidence that activation of epidermal growth factor receptor signalling pathways promotes GLT-1 expression (Zelenaia et al., 2000).
The 5' end of the rodent GLT-1 gene has not been published to date, however. The regulatory elements determining GLT-1 expression are therefore unknown. Observ ations in astroglial cultures suggest that the nuclear transcription factor NF-
B participates in the regulation of GLT-1 expression (Zelenaia et al., 2000). Further studies are required for a more complete understanding of the regulation of GLT-1 expression.
Further evidence for glial dysfunction in R6/2 mice: alterations of the glutamate–glutamine cycle and of taurine metabolism.
Glutamine–glutamate cycle
We showed a substantial increase of extracellular glutamine levels in aging R6/2 mice, indicating a perturbed glutamate–glutamine cycling (Daikhin and Yudkoff, 2000). It is well established that the brunt of glutamate released from glutamatergic neurones is taken up by a subpopulation of astrocytes via GLT-1. Glial cells convert glutamate into glutamine via the GS pathway. This step requires ammonia derived from blood or brain metabolism. Glutamine in turn is released from glia cells and enters the extracellular compartment by passive diffusion. Extracellular glutamine is taken up into the neuronal compartment by both sodium-dependent and -independent pathways. Intraneuronal glutamine is hydrolysed by PAG to glutamate and ammonia in mitochondria. A fraction of glutamate derived from glutamine is used to replenish the neurotransmitter pool, but the majority is oxidized to meet neuronal energy requirements (Daikhin and Yudkoff, 2000).
An increase in extracellular glutamine levels may therefore result from an increased production of glutamine in glial cells, enhancing the release of glutamine into the extracellular compartment, or it may reflect decreased uptake into neurones.
The finding of normal GS protein levels does not at first glance suggest that increased extracellular glutamine levels primarily reflect an increased production of glutamine. However, an increased glutamine content has been reported in brains of R6/2 animals (Jenkins et al., 2000), consistent with an increased production or an impaired breakdown of glutamine. This finding in R6/2 mice is partly at odds with observations in Huntington’s disease post-mortem brain demonstrating normal glutamine content (Perry, 1981), although an increased glutamine content was observed in the putamen of Huntington’s disease brains (Perry, 1981). Therefore, glutamine production may not be uniformly altered in Huntington’s disease: in severely affected areas, populations of glial cells with upregulated glutamine production may predominate.
Indeed we observed that individual glial cells in R6/2 mice displayed enlarged cell bodies and extended processes, which were densely stained by the GS antibody. This suggests an upregulation of GS in a subset of glial cells, despite a decreased density of GS-immunoreactive cells overall. The overall decrease in the density of GS-immunoreactive cells possibly accounts for the normal GS protein levels in homogenates, and is consistent with an overall reduction in GS activity in Huntington’s disease brains in all regions studied (Carter, 1981).
It is unlikely that the diabetic profile in R6/2 mice accounts for the increase in extracellular glutamine because studies of glutamine concentrations showed normal levels in brain (Makar et al., 1995) and in plasma (Walsh et al., 1998) of diabetic humans and animals. Similarly, there is no evidence for increased levels of ammonia in R6/2 brain, which are known to elevate brain glutamine levels.
Increased extracellular glutamine levels may reflect impaired neuronal glutamine uptake. We did not study glutamine transport. However, increased tissue content of glutamine in R6/2 argues for either increased production or impaired degradation rather than a primary transport deficit.
Glutamine degradation may be impaired by a decreased activity of the neuronal PAG. We found no change in the protein expression or immunohistochemical staining pattern of PAG in R6/2 brains. This does not exclude a reduction of enzymatic activity, and clearly PAG activity in R6/2 brains deserves further study. Interestingly, in the caudate nucleus of Huntington’s disease patients, reduced PAG activity was found (Butterworth et al., 1985). It is unclear at present whether the reduced PAG activity in human caudate but not in other brain regions reflects neuronal loss or mitochondrial dysfunction (Browne et al., 1997; Tabrizi et al., 1999) preceding neuronal loss.
Taurine
We observed a marked progressive elevation of extracellular taurine levels in R6/2 mice compared with control littermates. A similarly substantial increase in brain tissue content of taurine in R6/2 was reported recently (Jenkins et al., 2000). This suggests that an increased formation or an impaired degradation of taurine, rather than an enhanced release into the extracellular compartment, e.g. by reversal of the taurine transporter (Huxtable, 1989; Scheller et al., 2000), accounts for our findings. It is proposed that taurine is synthesized preferentially by astrocytes (Reymond et al., 1996); however, the conditions that induce its formation are not well understood. Taurine has been shown to be released in large concentrations during ischaemia and excitotoxin-induced acute neuronal damage (Huxtable, 1989; Scheller et al., 2000) and may exert a protective effect (Huxtable, 1989), indicating regulation of taurine levels by glial–neuronal interactions. Taken together, the marked increase of extracellular taurine levels as well as of brain taurine content support the notion that a glial cell dysfunction is part of the pathophysiology of Huntington’s disease. The downstream effects of the Huntington’s disease mutation affect glial cell functions relatively early, given that glial GLT-1 expression is already reduced at 6 weeks of age in R6/2 mice and that glutamate–glutamine cycling starts to be perturbed at a similar time point.
Implications for the pathogenesis of Huntington’s disease
The apparent downregulation of GLT-1 in Huntington’s disease brain raises the possibility that elevated extracellular glutamate levels may contribute to neuronal loss via excitotoxic mechanisms. However, neuronal loss in R6/2 mice is rare despite major functional derangements. In addition, R6/2 mice exposed to pulses of PDC resulting in elevated extracellular glutamate concentrations for a short time period did not display frank neuronal loss, in good agreement with the effects of single intrastriatal injections of PDC in normal animals (Massieu and Tapia, 1997). It is nevertheless tempting to speculate that a downregulation of glutamate transport for a prolonged period may eventually result in elevated synaptic glutamate levels and neuronal loss by perisynaptic dysregulation (Lievens et al., 2000). The small number of neurones undergoing unequivocal cell death in the R6/2 transgenic Huntington’s disease model (Turmaine et al., 2000) may reflect the fact that for the lifetime of these animals, extracellular glutamate levels can, by and large, be maintained within the normal range, despite a major reduction in transport capacity. Neuronal death should ensue once the limits of functional compensation are exceeded and extracellular glutamate levels are persistently elevated. Studies in transgenic models of Huntington’s disease displaying frank neuronal loss are currently under way to test this hypothesis.
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TopSummaryIntroductionMaterials and methodsResultsDiscussionConclusionsReferences |
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We provide evidence for an in vivo impairment of glutamate handling in a transgenic mouse model of Huntington’s disease, which begins before the onset of symptoms in the animals. These findings support the view that excitotoxic mechanisms play a role in the pathogenesis of Huntington’s disease and add Huntington’s disease to the list of neurodegenerative disorders associated with compromised glutamate transport capacity. Clearly further studies are required to elucidate the mechanisms of GLT-1 dysfunction. However, our findings in R6/2 mice, if confirmed in other transgenic models and in Huntington’s disease brain, suggest that compounds reducing extracellular glutamate concentrations or increasing GLT-1 expression and function have a potential for modifying the relentlessly progressive course of Huntington’s disease.
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Acknowledgements |
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We wish to thank G. Bates (Guy’s and St Thomas’ Hospital, London, UK) for providing the mice, A. Mahal (Guy’s and St Thomas’ Hospital, London, UK) and B. Zucker (Freiburg Universitatsklinik, Germany) for genotyping the animals, I. Sillaber (Max Planck Institut für Psychiatrie München, Germany) for aid in establishing the microdialysis in mice, and R. Rossner (Dipl. Math., Freiburg) for statistical analysis of the data. We also wish to thank B. Schwalenstöcker and T. Meyer (University of Ulm, Germany) for providing a template for the GLT-1 probe, J. D. Rothstein (Johns Hopkins University, Baltimore, MD, USA) and N. Curthoys (Colorado State University, Fort Collins, CO, USA) for providing antibodies, and H. Langemann (Kantonsspital, Basel, Switzerland) for careful reading of the manuscript and helpful discussions. This work was supported by Deutsche Forschungsgemeinschaft (SFB 505, TP C2) and by the Wellcome Trust.
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