Three glutamate transporters have been identified in rat, including astroglial transporters GLAST and GLT-1 and a neuronal transporter EAAC1. Here we demonstrate that inhibition of the synthesis of each glutamate transporter subtype using chronic antisense oligonucleotide administration, in vitro and in vivo, selectively and specifically reduced the protein expression and function of glutamate transporters. The loss of glial glutamate transporters GLAST or GLT-1 produced elevated extracellular glutamate levels, neurodegeneration characteristic of excitotoxicity, and a progressive paralysis. The loss of the neuronal glutamate transporter EAAC1 did not elevate extracellular glutamate in the striatum but did produce mild neurotoxicity and resulted in epilepsy. These studies suggest that glial glutamate transporters provide the majority of functional glutamate transport and are essential for maintaining low extracellular glutamate and for preventing chronic glutamate neurotoxicity.

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Summary Oligodendroglia support axon survival and function through mechanisms independent of myelination and their dysfunction leads to axon degeneration in several diseases. The cause of this degeneration has not been determined, but lack of energy metabolites such as glucose or lactate has been hypothesized. Lactate is transported exclusively by monocarboxylate transporters, and changes to these transporters alter lactate production and utilization. We show the most abundant lactate transporter in the CNS, monocarboxylate transporter 1 (MCT1), is highly enriched within oligodendroglia and that disruption of this transporter produces axon damage and neuron loss in animal and cell culture models. In addition, this same transporter is reduced in patients with, and mouse models of, amyotrophic lateral sclerosis (ALS), suggesting a role for oligodendroglial MCT1 in pathogenesis. The role of oligodendroglia in axon function and neuron survival has been elusive; this study defines a new fundamental mechanism by which oligodendroglia support neurons and axons.

Glutamate is the principal excitatory neurotransmitter in the nervous system. Inactivation of synaptic glutamate is handled by the glutamate transporter GLT1 (also known as EAAT2; refs 1, 2), the physiologically dominant astroglial protein. In spite of its critical importance in normal and abnormal synaptic activity, no practical pharmaceutical can positively modulate this protein. Animal studies show that the protein is important for normal excitatory synaptic transmission, while its dysfunction is implicated in acute and chronic neurological disorders, including amyotrophic lateral sclerosis (ALS), stroke, brain tumours and epilepsy. Using a blinded screen of 1,040 FDA-approved drugs and nutritionals, we discovered that many beta-lactam antibiotics are potent stimulators of GLT1 expression. Furthermore, this action appears to be mediated through increased transcription of the GLT1 gene. beta-Lactams and various semi-synthetic derivatives are potent antibiotics that act to inhibit bacterial synthetic pathways. When delivered to animals, the beta-lactam ceftriaxone increased both brain expression of GLT1 and its biochemical and functional activity. Glutamate transporters are important in preventing glutamate neurotoxicity. Ceftriaxone was neuroprotective in vitro when used in models of ischaemic injury and motor neuron degeneration, both based in part on glutamate toxicity. When used in an animal model of the fatal disease ALS, the drug delayed loss of neurons and muscle strength, and increased mouse survival. Thus these studies provide a class of potential neurotherapeutics that act to modulate the expression of glutamate neurotransmitter transporters via gene activation.

ABSTRACTprior to the addition of drugs. To produce chronic toxicity, long-term inhibition of glutamate uptake was effected by incubating slices with culture medium containing various concentrations of THA or PDC (5, 6). At very high concentrations (>1 mM), THA can have weak actions at the N-methyl-D-aspartate (NMDA) receptor, a property not shared by PDC (5). Transport inhibitors were maintained in cultures by replenishing them at each change in culture medium. In all experiments, potentially neuroprotective drugs were added repeatedly, either in the presence or absence of a glutamate transport inhibitor, again beginning after 8 days in culture. Motor-neuron toxicity was monitored by two methods: (i) biochemical analysis of tissue choline acetyltransferase activity (ChAT) and (ii) microscopic morphology. ChAT activity is largely restricted to ventral motor neurons in rat lumbar spinal cord, and assays of ChAT activity have been used as a reliable marker for motor neurons (7-9). Motor neurons were also visualized in organotypic cultures by histological analysis of stained semithin plastic sections and by immunohistochemistry.Organotypic Spinal Cord Cultures. Organotypic spinal cord cultures were prepared using lumbar spinal cord slices from 8-day-old rat pups. Neonatal rat pups were decapitated, and the spinal cords were rapidly harvested and cultured by a modification of described methods (10, 11). Lumbar spinal cords were collected under sterile conditions and sectioned transversely at 350-,um intervals with a McIlwain tissue chopper (Mickle Laboratory Engineering, Gomshall, Surrey, U.K.). Sections were then transferred to sterile Gey's balanced salt solution (GIBCO) containing glucose (6.4 mg/ml) and gently separated at room temperature. Slices were carefully placed on the surface of 30-mm Millipore Millicell-CM porous (0.4 ,um) membranes (five slices per membrane). Such tissue grows optimally at an air/fluid interface, so it was important to remove any excess medium on the membrane surface around slices. The membranes were placed in 35-mm culture wells (Nunc) containing 1 ml of incubation medium [50% (vol/vol) minimal essential medium-25 mM Hepes/ 25% (vol/vol) heat-inactivated horse serum/25% (vol/vol) Hanks' balanced salt solution (GIBCO) supplemented with D-glucose (25.6 mg/ml) and glutamine (2 mM), at a final pH of 7.2]. Initial studies using pH 7.4 or 7.8 revealed similar results. Antibiotic and antifungal agents were not used.Cultures were incubated at 37°C in a 5% C02/95% air humidified environment (Forma Scientific, Marietta, OH). Culture medium, along with any added pharmacological agents, was changed twice weekly. By using this technique, >95% of the explants can be maintained in culture for >3 months with excellent organotypic cellular organization.