Purpose of Study Periventricular leukomalacia is a pathological condition affecting developing oligodendrocytes. Developing oligodendrocytes are vulnerable to injury due to oxidative stress such as that caused by high extracellular glutamate. Glutamate is an excitotoxic amino acid. Glutamate transporters are used by many cells in the nervous system to regulate extracellular glutamate levels. To date no studies have investigated the mechanism by which oligodendrocyte precursors process glutamate. It is unclear if it is by the classic EAAT pathway or by the cystine/glutamate exchanger. Thus, the aim of this study is to help characterize glutamate transporters in oligodendrocyte precursors.
Methods Mouse embryonic stem cells were ordered from American Tissue Culture Company (ATCC) and placed on a layer of 0.1% gelatin. Cells were grown for 5 days in the presence of 1x-beta-mercaptoethanol, 1x-leukemia inhibitory factor, and ES cell media (5% fetal bovine serum, 5% newborn calf serum). Cells were then trypsinized and plated again on 0.1% gelatin and treated with 5 micromolar cytosine arabinofuranoside (Ara-C). Cells were used for either RNA or immunocytochemistry. RT-PCR was performed using a Qiagen One-Step PCR kit. Routine protocols were used for immunocytochemistry. Forward and reverse primers were designed for the mouse cystine/glutamate exchanger, EAAT 1 and 2, and actin (control). Antibodies used for immunocytochemistry of oligodendrocytes were O1, O4, and NG2. CD98 antibody was used to recognize the amino acid transporter.
Results Oligodendrocyte precursors possess the cystine/glutamate exchanger as determined by immunocytochemistry and RT-PCR. EAAT 1 and 2 are present in the oligodendrocyte precursors as determined by RT-PCR.
Conclusions The EAATs are present in oligodendrocyte precursors. Oligodendrocyte precursors have the messenger RNA for the cystine/glutamate exchanger. The immunocytochemistry data suggest that the cystine/glutamate exchanger protein is also present. Future studies are under way to investigate the functionality of this exchanger in oligodendrocyte precursors and its role in glutamate metabolism.
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