Expression of a Variant Form of the Glutamate Transporter GLT1 in Neuronal Cultures and in Neurons and Astrocytes in the Rat Brain

Abstract

To identify glutamate transporters expressed in forebrain neurons, we prepared a cDNA library from rat forebrain neuronal cultures, previously shown to transport glutamate with high affinity and capacity. Using this library, we cloned two forms, varying in the C terminus, of the glutamate transporter GLT1. This transporter was previously found to be localized exclusively in astrocytes in the normal mature brain. Specific antibodies against the C-terminal peptides were used to show that forebrain neurons in culture express both GLT1a and GLT1b proteins. The pharmacological properties of glutamate transport mediated by GLT1a and GLT1b expressed in COS-7 cells and in neuronal cultures were indistinguishable. Both GLT1a and GLT1b were upregulated in astrocyte cultures by exposure to dibutyryl cAMP. We next investigated the expression of GLT1b in vivo. Northern blot analysis of forebrain RNA revealed two transcripts of ϳ3 and 11 kb that became more plentiful with developmental age. Immunoblot analysis showed high levels of expression in the cortex, hippocampus, striatum, thalamus, and midbrain. Pre-embedding electron microscopic immunocytochemistry with silver-enhanced immunogold detection was used to localize GLT1b in vivo. In the rat somatosensory cortex, GLT1b was clearly expressed in neurons in presynaptic terminals and dendritic shafts, as well as in astrocytes. The presence of GLT1b in neurons may offer a partial explanation for the observed uptake of glutamate by presynaptic terminals, for the preservation of input specificity at excitatory synapses, and may play a role in the pathophysiology of excitotoxicity.

EAAT2 regulation and splicing: relevance to psychiatric and neurological disorders

“…23 The first real efforts to characterize GLT1b occurred after two independent groups cloned the transcript from rat neurons. 24,25 This variant included the N-terminal region of the original isoform cloned by Pines and colleagues with the C-terminus identified previously in liver and brain. The 11 unique amino acids at the C-terminus of GLT1b encode a PDZ-binding domain that may allow this isoform to interact with synaptic scaffolding molecules.…”

“…The regional expression profiles of the GLT1a and GLT1b proteins are very similar, with high expression in the cortex, hippocampus, striatum, and thalamus and lower expression in the cerebellum, pons, and medulla. 24 The transport properties of the two splice Figure 1 Diagram of glutamate transporter 1 (GLT1) Cterminal splice variants. Exons 10 and 11 are shown and the unique exons of GLT1b and GLT1c are depicted as narrow boxes.…”

“…The sequence that makes up exon 11 in GLT1 is part of the 3 0 -UTR of GLT1b and GLT1c. 24,26 suggesting that any functional difference between the two isoforms likely involves different cellular localization patterns or participation in different signaling pathways. A third functional GLT1 variant encoding yet another novel C-terminus, referred to as GLT1c, was recently identified in the rat and human retina.…”

“…Both probes produced a signal at B11 kb and the GLT1b probe also produced a robust band at B3 kb. 24 Another northern blotting experiment, using RNA from rat whole brain, hippocampus and neocortex with probes that recognized GLT1 and GLT1b, identified an 11 kb transcript for GLT1 and a 12.5 kb transcript for GLT1b. 25 However, the authors of these studies did not discuss the longer GLT1b transcript or attempt to account for the additional sequence.…”

“…24,25 As neither group had previously been able to detect the main GLT1 isoform in neurons, as GLT1 immunoreactivity in the retina is confined to neurons, 52 and because GLT1b is the major isoform in the retina, 53 it seemed plausible at the time that GLT1b might be a primarily neuronal variant. However, more recent studies have established that GLT1 is far more abundant than GLT1b in both neurons and astrocytes, indicating that GLT1b is neither unique to neurons, nor is it the predominant isoform in neurons.…”