Reciprocal communication between astrocytes and endothelial cells is required for astrocytic glutamate transporter 1 (GLT-1) expression

Astrocytes have diverse functions that are supported by their anatomic localization between neurons and blood vessels. One of these functions is the clearance of extracellular glutamate. Astrocytes clear glutamate using two Na+-dependent glutamate transporters, GLT-1 (also called EAAT2) and GLAST (also called EAAT1). GLT-1 ex pression increases during synaptogenesis and is a marker of astrocyte maturation. Over 20 years ago, several groups demonstrated that astrocytes in culture express little or no GLT-1 and that neurons induce expression. We recently demonstrated that co-culturing endothelia with mouse astrocytes also induced expression of GLT-1 and GLAST. These increases were blocked by an inhibitor of gamma-secretase. This and other observations are consistent with the hypothesis that Notch signaling is required, but the ligands involved were not identified. In the present study, we used rat astrocyte cultures to further define the mechanisms by which endothelia induce expression of GLT-1 and GLAST. We found that co-cultures of astrocytes and endothelia express higher levels of GLT-1 and GLAST protein and mRNA. That endothelia activate Hes5, a transcription factor target of Notch, in astrocytes. Using recombinant Notch ligands, anti-Notch ligand neutralizing antibodies, and shRNAs, we provide evidence that both Dll1 and Dll4 contribute to endothelia-dependent regulation of GLT-1. We also provide evidence that astrocytes secrete a factor(s) that induces expression of Dll4 in endothelia and that this effect is required for Notch-dependent induction of GLT-1. Together these studies indicate that reciprocal communication between astrocytes and endothelia is required for appropriate astrocyte maturation and that endothelia likely deploy additional non-Notch signals to induce GLT-1.