The acidic amino acids, glutamate (Glu) and aspartate, are the predominant excitatory neurotransmitters in the mammalian central nervous system (CNS). Although there are millimolar concentrations of these excitatory amino acids (EAAs) in brain, extracellular concentrations are maintained in the low micromolar range to facilitate crisp synaptic transmission and to limit the neurotoxic potential of these EAAs. A family of Na+-dependent high affinity transporters is responsible for the regulation and clearance of extracellular EAAs. This project represents a collaborative effort between two laboratories that have defined the biochemical, pharmacological, and pathophysiological properties of these transporters. In this competitive renewal, we propose to focus on studies of one of the astroglial transporters, GLT-1/EAAT2. There is a substantial body of evidence to suggest that this transporter mediates the largest percentage of transport activity in the mammalian forebrain. The expression (protein and mRNA) of this transporter is regulated in several interesting ways, increasing dramatically during the period of synaptogenesis and astrocyte differentiation. Unlike the two other forebrain transporters, expression of GLT-1 is largely restricted to the CNS and astrocytic expression is dependent upon the presence of neurons both in vivo and in vitro. Finally, we and others have documented abnormal expression of this transporter in several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Essentially nothing is known about the mechanisms responsible for this regulation of GLT-1. Therefore in Specific Aims I-III, we propose a strategy to study the mechanisms that control GLT-1 expression and assembly. We and others have shown that inhibition of these transporters increases the susceptibility of the tissue to excitotoxic insults, but it is not known if overexpression can be neuroprotective. Therefore in Specific Aim IV, we propose to determine if over-expression of GLT-1 is neuroprotective in animals models of ALS and against excitotoxic insults using both in vitro and in vivo models.
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