My long-term goals are to investigate whether subtype-specific regulation of glutamate transporter gene expression during periods of neuronal insult associated with ischemia, anoxia, epilepsy, amyotrophic lateral sclerosis and psychiatric disorders such as schizophrenia results in excitotoxic cell death or is a mechanism for neuroprotection against increased glutamate efflux via the glutamate transporter. Clearance of excitatory amino acid neurotransmitters from the synaptic cleft occurs through diffusion and reuptake of glutamate into presynaptic terminals and surrounding glial cells via high-affinity neuronal and glial glutamate transporters. Through the use of uptake blockers it has been demonstrated that excitatory amino acid transporter (EAAT) function is essential for the maintenance of extracellular glutamate concentrations below neurotoxic levels. It would therefore appear that reduced expression of the principal subtype of glutamate transporter, EAAT2, under CNS insult would be a mechanism for excitotoxic cell death. However, glutamate uptake via excitatory amino acid transporters is driven by the movement of K+ and Na+ ions along their electrochemical gradients, and reversal of K+ and Na+ ion gradients following ischemia or hypoxia can therefore result in glutamate efflux via reversal of the glutamate transporter. Hence reduced expression of EAAT2 may be neuroprotective under these conditions. To specifically address the consequences of decreased EAAT2 glutamate transporter gene expression under hypoxic conditions, I have developed a new transgenic model of EAAT2 overexpression, which results in (1) astrocyte-specific increases in the EAAT2 transgene expression and (2) further induction of transgene expression under hypoxic conditions. If down-regulation of EAAT2 is neuroprotective due to a decrease in glutamate efflux, then in the EAAT2 transgenic model, where more EAAT2 protein is made under hypoxic conditions, this increase in EAAT2 protein should lead to an increase in glutamate efflux and hence increased excitotoxic cell death. To test the hypothesis that down-regulation of EAAT2 gene expression in astrocytes is a primary event leading to neuroprotection of surrounding neurons, the phenotype of the EAAT2 transgenics will be fully characterized using in situ hybridization, RNase protection, immunochemistry, histology and synaptosomal uptake analysis prior to using the EAAT2 transgenics in an vivo analysis of excitotoxic cell death under normal and hypoxic conditions (Specific Aim 1). Primary cortical cultures from wild type (+/+) and EAAT2 transgenic mice will be used to quantitate changes in neuronal survival under normal and hypoxic conditions (Specific Aim 2).
