A function specific to astrocytes at the excitatory synapse is clearance of excess glutamate. We therefore investigated the effect of NHE9 and its variants on glutamate uptake in astrocytes. GLAST (GLutamate ASpartate Transporter) is a high-affinity, Na+-dependent glutamate transporter highly expressed in astrocytes46 where it partially co-localizes with endosomal NHE9 (Figure 9A–D). Overexpression of NHE9 resulted in ~1.9 fold increase in 3H-glutamate uptake relative to control cells, whereas all three autism-associated variants were similar to vector-transformed control (Figure 9E). Although total amounts remained unchanged in all cell lines, surface expression of GLAST transporter increased by ~2 fold in astrocytes expressing wild type NHE9 but not the autism-associated variants L236S, S438P and V176I (Figure 9 F–G). Consistent with these observations, alkalinization of the transferrin-positive endosomal compartment was only observed in cells expressing wild type NHE9 (Figure 10A–B). Taken together, our findings indicate that all three autism-associated variants were associated with loss of function phenotypes in astrocytes.
