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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The pathogenesis of sporadic amyotrophic lateral sclerosis (ALS) is unknown, but defects in synaptosomal high-affinity glutamate transport have been observed. In experimental models, chronic loss of glutamate transport can produce a loss of motor neurons and, therefore, could contribute to the disease. With the recent cloning of three glutamate transporters, i.e., EAAC1, GLT-1, and GLAST, it has become possible to determine if the loss of glutamate transport in ALS is subtype specific. We developed C-terminal, antioligopeptide antibodies that were specific for each glutamate transporter. EAAC1 is selective for neurons, while GLT-1 and GLAST are selective for astroglia. Tissue from various brain regions of ALS patients and controls were examined by immunoblot or immunocytochemical methods for each transporter subtype. All tissue was matched for age and postmortem delay. GLT-1 immunoreactive protein was severely decreased in ALS, both in motor cortex (71% decrease compared with control) and in spinal cord. In approximately a quarter of the ALS motor cortex specimens, the loss of GLT-1 protein (90% decrease from control) was dramatic. By contrast, there was only a modest loss (20% decrease from control) of immunoreactive protein EAAC1 in ALS motor cortex, and there was no appreciable change in GLAST. The minor loss of EAAC1 could be secondary to loss of cortical motor neurons. As a comparison, glial fibrillary acidic protein, which is selectively localized to astroglia, was not changed in ALS motor cortex. Because there is no loss of astroglia in ALS, the dramatic abnormalities in GLT-1 could reflect a primary defect in GLT-1 protein, a secondary loss due to down regulation, or other toxic processes.

ALS is associated with a defect in high-affinity glutamate transport that has disease, region, and chemical specificity. Defects in the clearance of extracellular glutamate because of a faulty transporter could lead to neurotoxic levels of extracellular glutamate and thus be pathogenic in ALS.

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.

Recently, the excitatory amino acid neurotransmitter glutamate was implicated in the pathogenesis of a variety of chronic degenerative neurological diseases in humans and animals. This report describes abnormalities in excitatory amino acids in the central nervous system of 18 patients with amyotrophic lateral sclerosis (ALS). The concentration of the excitatory amino acids glutamate and aspartate in the cerebrospinal fluid were increased significantly (p less than 0.01) by 100 to 200% in patients with ALS. Similarly, the concentrations of the excitatory neuropeptide N-acetyl-aspartyl glutamate and its metabolite, N-acetyl-aspartate, were elevated twofold to threefold in the cerebrospinal fluid from the patients. There was no relationship between amino acid concentrations and duration of disease, clinical impairment, or patient age. In the ventral horns of the cervical region of the spinal cord, the level of N-acetyl-aspartyl glutamate and N-acetyl-aspartate was decreased by 60% (p less than 0.05) and 40% (p less than 0.05), respectively, in 8 patients with ALS. Choline acetyltransferase activity was also diminished by 35% in the ventral horn consistent with motor neuron loss. We conclude that excitatory amino acid metabolism is altered in patients with ALS. Based on neurodegenerative disease models, these changes may play a role in motor neuron loss in ALS.

Although colchicine has been used for centuries, its neuromuscular toxicity in humans is largely unrecognized. In this report we describe a characteristic syndrome of myopathy and neuropathy and present 12 new cases of the condition. Colchicine myopathy may occur in patients with gout who take customary doses of the drug but who have elevated plasma drug levels because of altered renal function. It usually presents with proximal weakness and always presents with elevation of serum creatine kinase; both features remit within three to four weeks after the drug is discontinued. The accompanying axonal polyneuropathy is mild and resolves slowly. Electromyography of proximal muscles shows a myopathy that is marked by abnormal spontaneous activity. Because of these features, colchicine myoneuropathy is usually misdiagnosed initially, either as probable polymyositis or as uremic neuropathy. The myopathy is vacuolar, marked by accumulation of lysosomes and autophagic vacuoles unrelated to necrosis or to the mild denervation in distal muscles. The morphologic changes in muscle suggest that the pathogenesis involves disruption of a microtubule-dependent cytoskeletal network that interacts with lysosomes. Correct diagnosis may save patients with this disorder from inappropriate therapy.

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