Astrocytes are critical for the long-term modulation of neuronal synapses as well as acute clearance of the excitatory neurotransmitter, glutamate, from the synaptic cleft34. In animal models of autism, astrocytic clearance of glutamate is altered and glutamate transporter levels decreased44. Elevated levels of glutamate in the synapse trigger seizures, and seizures are well known to be co-morbid with autism. As shown by Morrow et al.29, a significant subset of NHE9 variants was associated with both autism and seizures. Furthermore, elevated brain glutamate levels are observed in patients with mutations in the closely related ortholog NHE619. Therefore, to confirm our findings in yeast and extend the analysis of autism-associated variants in NHE9 to a neurobiological model we chose to evaluate function in astrocytes19. We began by evaluating expression levels of NHE9 and NHE6 isoforms in primary mouse astrocytes. Transcript analysis revealed the presence of both NHE6 and NHE9 in cDNA extracts from astrocytes (Figure 5A) as well as in neurons (Figure 4D). Knockdown of NHE9 (by ~80%) in the astrocytes did not alter transcript levels for NHE6, although a modest compensatory
