Glutamate transporter expression and function in human glial progenitors.

Glutamate is the major neurotransmitter of the brain, whose extracellular levels are tightly controlled by glutamate transporters. Five glutamate transporters in the human brain (EAAT1-5) are present on both astroglia and neurons. We characterize the profile of three different human astroglial progenitors in vitro: human glial restricted precursors (HGRP), human astrocyte precursors (HAPC), and early-differentiated astrocytes. EAAT 1, EAAT3, and EAAT4 are all expressed in GRPs with a subsequent upregulation of EAAT1 following differentiation of GRPs into GRP-derived astrocytes in the presence of bone morphogenic protein (BMP-4). This corresponds to a significant increase in the glutamate transport capacity of these cells. EAAT2, the transporter responsible for the bulk of glutamate transport in the adult brain, is not expressed as a full-length protein, nor does it appear to have functional significance (as determined by the EAAT2 inhibitor dihydrokainate) in these precursors. A splice variant of EAAT2, termed EAAT2b, does appear to be present in low levels, however. EAAT3 and EAAT4 expression is reduced as glial maturation progresses both in astrocyte precursors and early-differentiated astrocytes and is consistent with their role in adult tissues as primarily neuronal glutamate transporters. These human glial precursors offer several advantages as tools for understanding glial biology because they can be passaged extensively in the presence of mitogens, afford the potential to study the temporal changes in glutamate transporter expression in a tightly controlled fashion, and are cultured in the absence of neuronal coculture, allowing for the independent study of astroglial biology.

Maragakis NJ ，Dietrich J ，Wong V ，Xue H ，Mayer-Proschel M ，Rao MS ，Rothstein JD ... -  《GLIA》

Neuronal glutamate transporter EAAT4 is expressed in astrocytes.

High-affinity excitatory amino acid transporters (EAATs) are essential to terminate glutamatergic neurotransmission and to prevent excitotoxicity. To date, five distinct EAATs have been cloned from animal and human tissues: GLAST (EAAT1), GLT-1 (EAAT2), EAAC1 (EAAT3), EAAT4, and EAAT5. EAAT1 and EAAT2 are commonly known as glial glutamate transporters, whereas EAAT3, EAAT4, and EAAT5 are neuronal. EAAT4 is largely expressed in cerebellar Purkinje cells. In this study, using immunohistochemistry and Western blotting, we found that EAAT4-like immunoreactivity (ir) is enriched in the spinal cord and forebrain. Double-labeled fluorescent immunostaining and confocal image analysis indicated that EAAT4-like ir colocalizes with an astrocytic marker, glial fibrillary acidic protein (GFAP). The astrocytic localization of EAAT4 was further confirmed in astrocyte cultures by double-labeled fluorescent immunocytochemistry and Western blotting. Reverse transcriptase-polymerase chain reaction analysis demonstrated mRNA expression of EAAT4 in astrocyte cultures. Sequencing confirmed the specificity of the amplified fragment. These results demonstrate that EAAT4 is expressed in astrocytes. This astrocytic localization of neuronal EAAT4 may reveal a new function of EAAT4 in the central nervous system.

Hu WH ，Walters WM ，Xia XM ，Karmally SA ，Bethea JR ... -  《GLIA》

Developmental expression of glutamate transporters and glutamate dehydrogenase in astrocytes of the postnatal rat hippocampus.

Glutamate is the major excitatory transmitter in the CNS and plays distinct roles in a number of developmental events. Its extracellular concentration, which mediates these activities, is regulated by glutamate transporters in glial cells and neurons. In the present study, we have used nonradioactive in situ hybridization, immunocytochemistry, and immunoblotting to show the cellular and regional expression of the high-affinity glutamate transporters GLAST (EAAT1) and generic GLT1 (EAAT2; glial form of GLT1) in the rat hippocampus during postnatal development (P1-60). The results of transporter expression were compared with the localization and activity pattern of glutamate dehydrogenase (GDH), an important glutamate-metabolizing enzyme. The study showed that both transporters and GDH were demonstrable at P1 (day of birth). The expression of GLAST (detected by in situ hybridization and immunocytochemistry) in the early postnatal development was higher than GLT1. Thereafter, the expression of both transporters increased, showing adult levels at between P20 and P30 (detected by in situ hybridization and immunoblotting). At these time points, the expression of GLT1 appeared to be significantly higher than the GLAST expression. GLT1 and GLAST proteins were demonstrable only in astrocytes. The increase of GDH activities (steepest increase from P5-P8), which were localized preferentially in astrocytes, was in agreement with the increase of transporter expression, preferentially with that of GLT1. These observations suggest that the extent of glutamate transporter expression and of glutamate-metabolizing GDH activity in astrocytes is intimately correlated with the formation of glutamatergic synapses in the developing hippocampus.

Kugler P ，Schleyer V 《HIPPOCAMPUS》

Loss of glial fibrillary acidic protein results in decreased glutamate transport and inhibition of PKA-induced EAAT2 cell surface trafficking.

Hughes EG ，Maguire JL ，McMinn MT ，Scholz RE ，Sutherland ML ... -  《-》

Localization and expression of the glutamate transporter, excitatory amino acid transporter 4, within astrocytes of the rat retina.

Ward MM ，Jobling AI ，Puthussery T ，Foster LE ，Fletcher EL ... -  《CELL AND TISSUE RESEARCH》