Functional and Transcriptional Downregulation of Glutamate Transporters (EAAT1 and EAAT2) via DNMT3B Overexpression in Glial Cells.

Glutamate (Glu) is the major excitatory neurotransmitter amino acid in the vertebrate brain. It elicits its actions through the activation of specific receptors in neurons and glial cells. Excitatory amino acid transporters 1 and 2 are the Glu transporters expressed in glial cells, responsible for recycling Glu and executing a protective role against excitotoxicity and neurodegeneration. In neurogenerative diseases and gliomas, glial cells fail to maintain Glu-glutamine homeostasis, triggering excitotoxicity and DNA methylation profile disruption. The aim of this contribution was to investigate the relationship between the methylation gained by DNA methylase 3B overexpression and the functional effect and the expression levels of Glu transporters in glial cell models. First, we confirmed the differential expression and deregulation of excitatory amino acid transporters 1 and 2 in cancer and neurodegenerative diseases. Next, Müller retinal cells were transfected with a DNA methylase 3B vector, and global 5-methylcytosine increased was detected after 48 h. Using a [3H]-D-Aspartate uptake assay in Müller glia cells and the tumoral U87 cells, we determined that de novo methylation affects the functional activity of the Glu transporters. The overexpression of DNMT3B also decreases the expression of Glu transporters in both cell models. Finally, due to the structural characteristics related to methylation deposition in DNA, we analyzed the methylation status of the excitatory amino acid 2 promoter, and we confirmed that DNA methylation increased in U87 cells by the DNMT3B overexpression. These findings suggest that de novo DNA methylation regulates the expression of glial Glu transporters and affects their functional role under excitotoxic environments. This work highlights the DNA methylation mechanism as a promising approach to understand the physiopathology landscape related to excitotoxic processes involved in brain diseases.