The potential role of glutamate transporters in the pathogenesis of normal tension glaucoma.

Glaucoma, a progressive optic neuropathy due to retinal ganglion cell (RGC) degeneration, is one of the leading causes of irreversible blindness. Although glaucoma is often associated with elevated intraocular pressure (IOP), IOP elevation is not detected in a significant subset of glaucomas, such as normal tension glaucoma (NTG). Moreover, in some glaucoma patients, significant IOP reduction does not prevent progression of the disease. Thus, understanding IOP-independent mechanisms of RGC loss is important. Here, we show that mice deficient in the glutamate transporters GLAST or EAAC1 demonstrate spontaneous RGC and optic nerve degeneration without elevated IOP. In GLAST-deficient mice, the glutathione level in Müller glia was decreased; administration of glutamate receptor blocker prevented RGC loss. In EAAC1-deficient mice, RGCs were more vulnerable to oxidative stress. These findings suggest that glutamate transporters are necessary both to prevent excitotoxic retinal damage and to synthesize glutathione, a major cellular antioxidant and tripeptide of glutamate, cysteine, and glycine. We believe these mice are the first animal models of NTG that offer a powerful system for investigating mechanisms of neurodegeneration in NTG and developing therapies directed at IOP-independent mechanisms of RGC loss.

Caenorhabditis elegans glutamate transporter deletion induces AMPA-receptor/adenylyl cyclase 9-dependent excitotoxicity.

In stroke and several neurodegenerative diseases, malfunction of glutamate (Glu) transporters causes Glu accumulation and triggers excitotoxicity. Many details on the cascade of events in the neurodegenerative process remain unclear. As molecular components of glutamatergic synapses are assembled in Caenorhabditis elegans and as many fundamental cellular processes are conserved from nematodes to humans, we studied Glu-induced necrosis in C. elegans and probed its genetic requirements. We combined deltaglt-3, a Glu transporter-null mutation, with expression of a constitutively active form of the alpha subunit of the G protein Gs. While neither deltaglt-3 nor expression of the constitutively active form of the alpha subunit of the G protein Gs is severely toxic to C. elegans head interneurons, their combination induces extensive neurodegeneration. deltaglt-3-dependent neurodegeneration acts through Ca2+-permeable Glu receptors of the alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid (AMPA) subtype, requires calreticulin function, and is modulated by calcineurin and type-9 adenylyl cyclase (AC9). We further show that mammalian AC9 hyperactivates mammalian AMPA-receptors (AMPA-Rs) in a Xenopus oocyte expression system, supporting that the relationship between AMPA-Rs hyperactivation and AC9 might be conserved between nematodes and mammals. AMPA-Rs-AC9 synergism is thus critical for nematode excitotoxicity and could potentially be involved in some forms of mammalian neurodegeneration.

Computational model of extracellular glutamate in the nucleus accumbens incorporates neuroadaptations by chronic cocaine.

Chronic cocaine administration causes instability in extracellular glutamate in the nucleus accumbens that is thought to contribute to the vulnerability to relapse. A computational framework was developed to model glutamate in the extracellular space, including synaptic and nonsynaptic glutamate release, glutamate elimination by glutamate transporters and diffusion, and negative feedback on synaptic release via metabotropic glutamate receptors (mGluR2/3). This framework was used to optimize the geometry of the glial sheath surrounding excitatory synapses, and by inserting physiological values, accounted for known stable extracellular, extrasynaptic concentrations of glutamate measured by microdialysis and glutamatergic tone on mGluR2/3. By using experimental values for cocaine-induced reductions in cystine-glutamate exchange and mGluR2/3 signaling, and by predicting the down-regulation of glutamate transporters, the computational model successfully represented the experimentally observed increase in glutamate that is seen in rats during cocaine-seeking. This model provides a mathematical framework for describing how pharmacological or pathological conditions influence glutamate transmission measured by microdialysis.

Altered hippocampal expression of glutamate receptors and transporters in GRM2 and GRM3 knockout mice.

Group II metabotropic glutamate receptors (mGluR2 and mGluR3, also called mGlu2 and mGlu3, encoded by GRM2 and GRM3, respectively) are therapeutic targets for several psychiatric disorders. GRM3 may also be a schizophrenia susceptibility gene. mGluR2-/- and mGluR3-/- mice provide the only unequivocal means to differentiate between these receptors, yet interpretation of in vivo findings may be complicated by secondary effects on expression of other genes. To address this issue, we examined the expression of NMDA receptor subunits (NR1, NR2A, NR2B) and glutamate transporters (EAAT1-3), as well as the remaining group II mGluR, in the hippocampus of mGluR2-/- and mGluR3-/- mice, compared with wild-type controls. mGluR2 mRNA was increased in mGluR3-/- mice, and vice versa. NR2A mRNA was increased in both knockout mice. EAAT1 (GLAST) mRNA and protein, and EAAT2 (GLT-1) protein, were reduced in mGluR3-/- mice, whereas EAAT3 (EAAC1) mRNA was decreased in mGluR2-/- mice. Transcripts for NR1 and NR2B were unchanged. The findings show a compensatory upregulation of the remaining group II metabotropic glutamate receptor in the knockout mice. Upregulation of NR2A expression suggests modified NMDA receptor signaling in mGluR2-/- and mGluR3-/- mice, and downregulation of glutamate transporter expression suggests a response to altered synaptic glutamate levels. The results show a mutual interplay between mGluR2 and mGluR3, and also provide a context in which to interpret behavioral and electrophysiological results in these mice.

Late appearance of glutamate transporter defects in a murine model of ALS-parkinsonism dementia complex.

Excitotoxicity has been widely hypothesized to play a major role in various neurodegenerative diseases. We have used a mouse model of ALS-parkinsonism dementia complex (ALS-PDC) of the Western Pacific to explore this hypothesis. Mice fed washed cycad flour, the major epidemiological link to ALS-PDC, showed significant and progressive motor, cognitive, and sensory behavioural deficits [Wilson, J.M., Khabazian, I., Wong, M.C., Seyedalikhani, A., Bains, J.S., Pasqualotto, B.A., Williams, D.E., Andersen, R.J., Simpson, R.J., Smith, R., Craig, U.K., Kurland, L.T., Shaw, C.A., 2002. Behavioral and neurological correlates of ALS-parkinsonism dementia complex in adult mice fed washed cycad flour. Neuromol. Med. 1 (3), 207-221]. In addition, glutamate transporter (GLT-1/EAAT2) levels measured by immunohistochemistry with antibodies specific for two glial glutamate transporter splice variants (GLT-1alpha and GLT-1B) were significantly down-regulated showing a 'patchy' loss of antibody label centered on blood vessels [Wilson, J.M., Khabazian, I., Pow, D.V., Craig, U.K., Shaw, C.A., 2003. Decrease in glial glutamate transporter variants and excitatory amino acid receptor down-regulation in a murine model of ALS-PDC. Neuromol. Med. 3 (2), 105-118]. Receptor binding assays showed decreased NMDA and AMPA receptor levels combined with increased GABA(A) receptor levels in various CNS regions. The alterations in GLT-1 variants and the ionotropic receptors are consistent with an increased level of extracellular glutamate. The interaction between environmental toxicity and genetic susceptibility was also tested using mice expressing various Apolipoprotein E (ApoE) genotypes. Mice lacking the ApoE gene showed relative resistance to cycad-induced toxicity as measured by GLT-1B labeling, but all mice expressing the human ApoE isoforms showed a similar loss of GLT-1B. We have further shown that an isolated cycad toxin (beta-sitosterol-beta-d-glucoside, BSSG), previously shown to release glutamate in vitro [Wilson, J.M., Khabazian, I., Wong, M.C., Seyedalikhani, A., Bains, J.S., Pasqualotto, B.A., Williams, D.E., Andersen, R.J., Simpson, R.J., Smith, R., Craig, U.K., Kurland, L.T., Shaw, C.A., 2002. Behavioral and neurological correlates of ALS-parkinsonism dementia complex in adult mice fed washed cycad flour. Neuromol. Med. 1 (3), 207-221], can be directly toxic to motor neurons in vivo [Wilson, J.M., Petrik, M.S., Moghadasian, M.H., Shaw, C.A., 2005. Examining the interaction of apo E and neurotoxicity on a murine model of ALS-PDC. Can. J. Physiol. Pharmacol. 83 (2), 131-141]. However, BSSG-fed mice did not show altered GLT-1B labeling in the spinal cord suggesting that an initial excitotoxic mechanism may not be responsible for the final neuronal loss observed. While glutamate-mediated excitotoxicity is likely involved in the outcomes following cycad/BSSG exposure, the precise location in the cascade of events ultimately leading to neuronal death remains to be determined.

Expression of glutamate transporters and ionotropic glutamate receptors in GLAST knockout mice.

In order to investigate the molecular mechanism underlying high seizure susceptibility of GLAST knockout mice, we carried out Western blotting for the expression of GLT-1, EAAC-1, and several kinds of glutamate receptors in the hippocampus and the cortex. Although no significant difference was observed between GLAST (+/+) and (-/-) mice in terms of expression of GLT-1 and EAAC-1 in the hippocampus, these proteins were over-expressed in the frontal cortex in GLAST (-/-) mice (GLT-1, about 210% increase; EAAC-1, about 180% increase). Expression of hippocampal Glu-R1 and Glu-R2 in GLAST (-/-) mice was remarkably increased (Glu-R1, about 140% increase; Glu-R2, about 160% increase), while Glu-R3 and NMDA receptors levels (NMDA-R1, 2A and 2B) were equal to those in control. Cortical levels of Glu-R1, -R2 and -R3 receptors in GLAST (-/-) mice were remarkably decreased (Glu-R1, about 60% decrease; Glu-R2, about 60% decrease; Glu-R3, about 70% decrease), while NMDA receptors were remarkably increased in comparison to those in GLAST (+/+) mice (N-R1, about 150% increase; N-R2A, about 150% increase; N-R2B, about 140% increase). These data suggest that the increased susceptibility to seizures in GLAST (-/-) mice might be derived from increased expression of Glu-R1 in the hippocampus coupled with decreased cortical expression of Glu-R2 and increased NMDA-R1 and -2A, -2B expression.

Contribution of a glial glutamate transporter to GABA synthesis in the retina.

Neuronal glutamate transporters have been shown to play a role in GABA synthesis by enhancing glutamate uptake. In the present study, we have examined whether a glial glutamate transporter, GLAST, has a role in GABA synthesis in the mammalian retina. We found that the retinal GABA level was about two-fold higher in the GLAST-/- mouse retina compared to that in the wild type. Endogenous glutamate level was also increased about 2-fold in the mutant. Therefore, loss of GLAST results in a higher retinal GABA level, probably due to increased availability of its precursor, glutamate. An increase in GABAergic activity can be expected to affect trigger features such as directional selective response of neurons in the GLAST-/- mouse retina.

Lack of glutamate transporter EAAC1 in the epididymis of infertile c-ros receptor tyrosine-kinase deficient mice.

Transgenic male mice carrying inactive mutations of the receptor tyrosine kinase c-ros lack the caput epididymidis initial segment and are infertile because sperm volume regulation is compromised. Complementary DNA arrays were used to detect differences in gene expression in the caput epididymidis of heterozygous fertile and homozygous infertile males. The glutamate transporter excitatory amino acid carrier 1 (EAAC1) was expressed in all epididymal regions with high expression in the initial segment and cauda epididymidis. Homozygous knockout mice did not express EAAC1 messenger RNA (mRNA) in the caput but they did express the gene in the corpus and cauda. Immunohistochemical staining for EAAC1 confirmed regional mRNA expression and demonstrated an adluminal location on stereocilia/microvilli of principal cells. The glutamate transporter-associated protein (GTRAP) 3-18 was detected in all epididymal regions independent of genotype, but a highly abundant novel transcript of 4.2 kilobases was found only in the initial segment of heterozygous c-ros mice. High-performance liquid chromatography measurement of glutamate revealed a significantly higher content in the proximal caput of infertile mice than fertile mice, and tissue glutamate content decreased distally in both genotypes. Because glutamate is used as an osmolyte in somatic cells, the lack of EAAC1 reported here may disturb normal osmolyte balance in the proximal epididymal lumen and compromise sperm maturation, in particular the development of sperm volume regulatory mechanisms.

Oxidative stress and disturbed glutamate transport in spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a hereditary motor neuron disease, and three clinical subtypes of autosomal recessive SMA, including Werdnig Hoffmann disease (type 1), have been shown to be induced by deletion within the same genes. In order to clarify the pathogenesis of motor neuron degeneration in SMA, we immunohistochemically examine the expressions of oxidative stress-related materials (oxidative products) and glutamate transporters, which can prevent glutamate neurotoxicity, in five autopsy cases of SMA type 1. Age-matched controls did not show any deposition of oxidative products in the brain. In contrast, the abnormal deposition of 4-hydroxy-2-nonenal-modified protein, a product of membrane lipid oxidation, was observed in the spinal motor neurons in three cases, although the motor neurons did not show an increase of nitrotyrosine, which was observed in adult-onset amyotrophic lateral sclerosis. In addition, the nuclei of neurons and glial cells in the precentral gyrus, thalamus or cerebellar cortex were immunoreactive for 8-hydroxy-2'-deoxyguanosine in two cases, which was one of the most commonly used markers for oxidative DNA damage. Regarding glial glutamate transporters, three of five cases of SMA type 1 showed a reduction in immunoreactivity for excitatory amino acid transporter-1 (GLAST) in the ventrolateral nucleus of the thalamus, in which there was neither neuronal loss nor gliosis in routine histochemistry. One case, having mechanical ventilation, demonstrated a reduced expression of another glial glutamate transporter (GLT-1) throughout the central nervous system. These data suggest that oxidative stress and disturbed glutamate transport can partly be involved in the motor neuron devastation and/or latent thalamic degeneration in SMA type 1.

[Role of glutamate transporters in astrocytes].

L-glutamate is the primary excitatory neurotransmitter in the mammalian central nervous system and has also been implicated as a potent neurotoxin. To ensure a high signal-to-noise ratio during synaptic transmission and to prevent neuronal damage that might occur as a result of excessive activation of glutamate receptors, the extracellular glutamate concentration is tightly controlled by glutamate transporters in the plasma membrane of neurons and the surrounding glial cells. Five subtypes of glutamate transporters have been identified and characterized by molecular cloning. Recent studies of glutamate transporters using the genetic knockout strategy indicate that glial, but not neuronal, glutamate transporters play critical roles in maintaining the extracellular glutamate concentrations and are thereby essential for both normal synaptic transmission and protection of neurons against glutamate excitotoxicity. Furthermore, we have recently demonstrated that glial glutamate transporters are necessary for brain development through regulation of extracellular glutamate concentration and mediate a functional metabolic crosstalk between neurons and astrocytes. Changes involving altered glutamate signaling are found in CNS disorders including depression, schizophrenia, and several neurodegenerative diseases. Therefore, the design of compounds capable of modulating glutamate uptake represents a novel strategy for the treatment of these CNS disorders.

Retinal glucose metabolism in mice lacking the L-glutamate/aspartate transporter.

The conventional view that glucose is the substrate for neuronal energy metabolism has been recently challenged by the "lactate shuttle" hypothesis in which glutamate cycling in glial cells drives all neuronal glucose metabolism. According to this view, glutamate released by activated retinal neurons is transported into Müller (glial) cells where it triggers glycolysis. The lactate released by Müller cells serves as the energy substrate for neuronal metabolism. Because the L-Glutamate/aspartate transporter (GLAST) is the predominant, Na+-dependent, glutamate transporter expressed by Müller cells, we have used GLAST-knockout (GLAST -/-) mice to examine the relationship between lactate release and GLAST activity in the retina. We found that glucose uptake and lactate production by the GLAST -/- mouse retina was similar to that observed in the wild type mouse retina. Furthermore, addition of 1 mM glutamate and NH4Cl to the incubation medium did not further stimulate glucose uptake in either case. When lactate release was measured in the presence of the lactate uptake inhibitor, alpha-cyano-4-hydroxycinnamate, there was no significant change in the amount of lactate released by retinas from GLAST -/- mice compared to the wild type. Finally, lactate release was similar under both dark and light conditions. These results show that lactate production and release is not altered in retinas of GLAST -/- mice, which suggests that metabolic coupling between photoreceptors and Müller cells is not mediated by the glial glutamate transporter, GLAST.

Decreased expression of glutamate transporters in genetic absence epilepsy rats before seizure occurrence.

In absence epilepsy, epileptogenic processes are suspected of involving an imbalance between GABAergic inhibition and glutamatergic excitation. Here, we describe alteration of the expression of glutamate transporters in rats with genetic absence (the Genetic Absence Epilepsy Rats from Strasbourg: GAERS). In these rats, epileptic discharges, recorded in the thalamo-cortical network, appear around 40 days after birth. In adult rats no alteration of the protein expression of the glutamate transporters was observed. In 30-day-old GAERS protein levels (quantified by western blot) were lower in the cortex by 21% and 35% for the glial transporters GLT1 and GLAST, respectively, and by 32% for the neuronal transporter EAAC1 in the thalamus compared to control rats. In addition, the expression and activity of GLAST were decreased by 50% in newborn GAERS cortical astrocytes grown in primary culture. The lack of modification of the protein levels of glutamatergic transporters in adult epileptic GAERS, in spite of mRNA variations (quantified by RT-PCR), suggests that they are not involved in the pathogeny of spike-and-wave discharges. In contrast, the alteration of glutamate transporter expression, observed before the establishment of epileptic discharges, could reflect an abnormal maturation of the glutamatergic neurone-glia circuitry.