Functional Evaluation of Autism Associated Mutations in Slc9A9 (NHE9)

A number of neurological disorders including autism, have recently been linked to mutations in a subgroup of intracellular Na+(K+)/H+ exchangers, which include NHE6 and NHE9. In this work, we examine the functional consequence of missense mutations in NHE9 associated with autism. In an attempt to determine mutations that are likely to actually be disease-causing amongst those variants identified by genetic studies, we used a unique homology modeling approach in combination with evolutionary analysis. We constructed a model for NHE9 using the NHE1 model as the structural template. Based on this analysis we chose mutations reported for the highly conserved L236, S438 and the moderately conserved V176 residues for further study. As a first approach, we used a yeast system to screen these NHE9 variants. Next, NHE9 mutations were expressed in mouse primary astrocytes for functional evaluation. NHE9 localized to early and recycling endosomes where it altered endosomal pH to effect the trafficking of glutamate transporter (GLAST) and consequently glutamate uptake. Further, we observed that this is not restricted to the astrocyte specific transporter as the uptake of transferrin by the transferrin receptor was also altered. Unlike the wild type protein, autism associated NHE9-mutants failed to show this effect. Taken together, these studies provide for the first time, a cellular assessment of the NEH9 mutations identified in autistic patients.