Commentary

Does excitotoxic cell death of motor neurons in ALS arise from glutamate transporter and glutamate receptor abnormalities?☆

Neural uptake of glutamate is executed by the structurally related members of the SLC1A family of solute transporters: GLAST/EAAT1, GLT-1/EAAT2, EAAC1/EAAT3, EAAT4, ASCT2. These plasma membrane proteins ensure supply of glutamate, aspartate and some neutral amino acids, including glutamine and cysteine, for synthetic, energetic and signaling purposes, whereas effective removal of glutamate from the synaptic cleft shapes excitatory neurotransmission and prevents glutamate toxicity. Glutamate transporters (GluTs) possess also receptor-like properties and can directly initiate signal transduction. GluTs are physically linked to other glutamate signaling-, transporting- and metabolizing molecules (e.g., glutamine transporters SNAT3 and ASCT2, glutamine synthetase, NMDA receptor, synaptic vesicles), as well as cellular machineries fueling the transmembrane transport of glutamate (e.g., ion gradient-generating Na/K-ATPase, glycolytic enzymes, mitochondrial membrane- and matrix proteins, glucose transporters). We designate this supramolecular functional assembly as ‘glutamosome’. GluTs play important roles in the molecular pathology of chronic pain, due to the predominantly glutamatergic nature of nociceptive signaling in the spinal cord. Down-regulation of GluTs often precedes or occurs simultaneously with development of pain hypersensitivity. Pharmacological inhibition or gene knock-down of spinal GluTs can induce/aggravate pain, whereas enhancing expression of GluTs by viral gene transfer can mitigate chronic pain. Thus, functional up-regulation of GluTs is turning into a prospective pharmacotherapeutic approach for the management of chronic pain. A number of novel positive pharmacological regulators of GluTs, incl. pyridazine derivatives and β-lactams, have recently been introduced. However, design and development of new analgesics based on this principle will require more precise knowledge of molecular mechanisms underlying physiological or aberrant functioning of the glutamate transport system in nociceptive circuits.

This article is part of the issue entitled ‘Special Issue on Neurotransmitter Transporters’.

Amyotrophic lateral sclerosis (ALS) is the most common adult-onset motor neuron disease characterized by a progressive degeneration of central and peripheral motor neurons, leading to the atrophy of voluntary muscles. It has been previously demonstrated that the kynurenine pathway (KP), the major biochemical pathway for tryptophan metabolism, is dysregulated in ALS. In particular, the neuroactive intermediate, quinolinic acid (QUIN) has been shown to accumulate with a concomitant decrease in other neuroprotective and immunomodulatory KP metabolites. Furthermore, multiple biochemical phenomena associated with QUIN cytotoxicity are present in ALS, suggesting that QUIN may play a substantial role in the pathogenesis of ALS. This review highlights the potential roles of QUIN in ALS, and explores KP modulation as a therapeutic candidate in ALS.

This article is part of the Special Issue entitled ‘The Kynurenine Pathway in Health and Disease’.

Glutamate is the predominant excitatory neurotransmitter in the mammalian CNS. It mediates essentially all rapid excitatory signaling. Dysfunction of glutamatergic signaling contributes to developmental, neurologic, and psychiatric diseases. Extracellular glutamate is cleared by a family of five Na⁺-dependent glutamate transporters. Two of these transporters (GLAST and GLT-1) are relatively selectively expressed in astrocytes. Other of these transporters (EAAC1) is expressed by neurons throughout the nervous system. Expression of the last two members of this family (EAAT4 and EAAT5) is almost exclusively restricted to specific populations of neurons in cerebellum and retina, respectively. In this review, we will discuss our current understanding of the mechanisms that control transcriptional regulation of the different members of this family. Over the last two decades, our understanding of the mechanisms that regulate expression of GLT-1 and GLAST has advanced considerably; several specific transcription factors, cis-elements, and epigenetic mechanisms have been identified. For the other members of the family, little or nothing is known about the mechanisms that control their transcription. It is assumed that by defining the mechanisms involved, we will advance our understanding of the events that result in cell-specific expression of these transporters and perhaps begin to define the mechanisms by which neurologic diseases are changing the biology of the cells that express these transporters. This approach might provide a pathway for developing new therapies for a wide range of essentially untreatable and devastating diseases that kill neurons by an excitotoxic mechanism.

Glutamate excitotoxicity is associated with a wide range of neurodegenerative disorders and also seems to be involved in the pathology of demyelinating disorders such as multiple sclerosis (MS). Cuprizone-induced toxic demyelination shows clear characteristics of MS such as demyelination and axonal damage without the involvement of the innate immune system. In this study, we have evaluated glutamate signaling during cuprizone-induced demyelination in the white and gray matter of mouse brain by studying the expression of ionotropic and metabotropic glutamate-receptors and -transporters by Affymetrix gene array analysis, followed by real-time PCR and western blot analysis. Cellular localization of glutamate transporters was investigated by fluorescence double-labeling experiments. Comparing white and gray matter areas, the expression of glutamate receptors was region-specific. Among NMDA receptor subunits, NR2A was up-regulated in the demyelinated corpus callosum (CC), whereas the metabotropic glutamate receptor mGluR2 was down-regulated in demyelinated gray matter. Glutamate–aspartate transporter (GLAST) co-localizing with GFAP⁺ astrocytes was increased in both demyelinated CC and telencephalic cortex, whereas Slc1a4 transporter was up-regulated only in CC. Our data indicate that cuprizone treatment affects glutamate-receptors and -transporters differently in gray and white matter brain areas revealing particularly regulation of GLAST and Slc1a4 compared with other genes. This might have an important influence on brain-region selective sensitivity to neurotoxic compounds and the progression of demyelination as has been reported for MS and other demyelinating neurological diseases.

This study aimed to evaluate the effect of riluzole on the activity of excitatory amino acid transporter type 3 (EAAT3), a neuronal glutamate transporter, and to investigate the role of protein kinase C (PKC) in this effect. EAAT3 expression was induced in Xenopus oocytes by injecting EAAT3 mRNA. Using the two-electrode voltage clamping method, membrane currents were recorded before, during, and after applying l-glutamate (30 μM) in the absence and presence of prior incubation with riluzole (0.3–100 μM). To study the effect of PKC on the riluzole-induced change in EAAT3 activity, oocytes were preincubated with 100 μM phorbol-12-myristate-13-acetate (PMA), a PKC activator, or PKC inhibitors (2 µM staurosporine and 100 µM chelerythrine) before the recording. Responses were quantified by integrating current traces and are reported in microCoulombs (μC). Riluzole reduced EAAT3 activity in a concentration-dependent manner (0.3–100 μM). Treatment of oocytes with PMA significantly increased the baseline and riluzole-reduced EAAT activity (P<0.05). In addition, treatment of oocytes with PKC inhibitors reduced basal transporter currents, but did not show a further significant decrease in the riluzole-reduced EAAT3 activity. These results suggest that riluzole reduces EAAT3 activity through PKC inhibition.

Drugs which upregulate astrocyte glutamate transport may be useful neuroprotective compounds by preventing excitotoxicity. We set up a new system to identify potential neuroprotective drugs which act through GLT-1. Primary mouse striatal astrocytes grown in the presence of the growth-factor supplement G5 express high levels of the functional glutamate transporter, GLT-1 (also known as EAAT2) as assessed by Western blotting and ³H-glutamate uptake assay, and levels decline following growth factor withdrawal. The GLT-1 transcriptional enhancer dexamethasone (0.1 or 1 μM) was able to prevent loss of GLT-1 levels and activity following growth factor withdrawal. In contrast, ceftriaxone, a compound previously reported to enhance GLT-1 expression, failed to regulate GLT-1 in this system. The neuroprotective compound riluzole (100 μM) upregulated GLT-1 levels and activity, through a mechanism that was not dependent on blockade of voltage-sensitive ion channels, since zonasimide (1 mM) did not regulate GLT-1. Finally, CDP-choline (10 μM–1 mM), a compound which promotes association of GLT-1/EAAT2 with lipid rafts was unable to prevent GLT-1 loss under these conditions. This observation extends the known pharmacological actions of riluzole, and suggests that this compound may exert its neuroprotective effects through an astrocyte-dependent mechanism.