Expression of the exon 9-skipping form of EAAT2 in astrocytes of rats.

mRNA for the exon 9-skipping form of the glutamate transporter excitatory amino acid transporter (EAAT) 2 (glutamate transporter 1, GLT-1) is known to be expressed in brain and spinal cord, and such expression was initially proposed to be associated with motor neuron disease. Surprisingly, a protein corresponding to the size of this splice variant has not previously been detected when using antibodies against one of the possible carboxyl terminal regions of EAAT2. This has been construed as indicating that little of the exon 9-skipping protein is expressed, or that such protein is not stable. We have now made selective antibodies against the splice site of this form of EAAT2. We show that in the adult rat brain and spinal cord, it is expressed primarily in populations of white matter astrocytes. Astrocytes expressing this splice variant also expressed glial fibrillary acidic protein. Expression was developmentally regulated, being expressed in a small number of astrocytes at postnatal day 7, but strongly expressed by large populations of white matter astrocytes by 25 days postnatum and into adulthood. Only a subset of gray matter astrocytes and radial glia expressed exon 9-skipping EAAT2. We suggest that exon 9-skipping EAAT2 may have a role in regulating extracellular glutamate in white matter tracts, either by interacting with normally spliced EAAT2 and modifying its targeting or transport activity, or by acting as a transporter itself. Conversely, the limited expression in gray matter suggests it is unlikely to be important for modulating synaptic levels of glutamate.

GLAST1b, the exon-9 skipping form of the glutamate-aspartate transporter EAAT1 is a sensitive marker of neuronal dysfunction in the hypoxic brain.

In normal brain, we previously demonstrated that the exon-9 skipping form of glutamate-aspartate transporter (GLAST; which we refer to as GLAST1b) is expressed by small populations of neurons that appear to be sick or dying and suggested that these cells were subject to inappropriate local glutamate-mediated excitation. To test this hypothesis we examined the expression of GLAST1b in the hypoxic pig brain. In this model glial glutamate transporters such as GLAST and glutamate transporter 1 (GLT-1) are down-regulated in susceptible regions, leading to regional loss of glutamate homeostasis and thus to brain damage. We demonstrate by immunohistochemistry that in those brain regions where astroglial glutamate transporters are lost, GLAST1b expression is induced in populations of neurons and to a lesser extent in some astrocytes. These neurons were also immunolabeled by antibodies against the carboxyl-terminal region of GLAST but did not label with antibodies directed against the amino-terminal region. Our Western blotting data indicate that GLAST1b expressed by neurons lacks the normal GLAST amino-terminal region and may be further cleaved to a smaller approximately 30-kDa fragment. We propose that GLAST1b represents a novel and sensitive marker for the detection of neurons at risk of dying in response to hypoxic and other excitotoxic insults and may have wider applicability in experimental and clinical contexts.