Autism constitutes a diverse set of disorders thought to affect neuronal connectivity and excitation:inhibition ratio (E:I). A 2008 study identified mutations in an endosomal sodium-proton exchanger, NHE9, in individuals with autism. Multiple groups have since demonstrated upregulation of NHE9 gene expression in postmortem autism brains, a result withstanding genome-wide corrections. Despite its apparent involvement in autism-related pathology, little is known about the function of NHE9 in development and circuitry. There is thus critical need to understand the role of endosomal NHEs in autism pathophysiology and potential novel treatments. The central hypothesis is that NHE9 regulates endosomal pH, governing trafficking and development of circuit function. The project includes three specific aims: Aim 1. To determine the function of NHE9 in regulating intra-endosomal pH and endosomal composition in neurons. The hypothesis is that NHE9 enables proton leak from the endosome, preventing over-acidification. Perturbing the level of NHE9 in neurons may also perturb the function of endosomes in the endocytic pathway. A pH-sensitive probe will be applied to primary neurons in culture, enabling quantification of pH levels in both absence and overexpression of NHE9. Aim 2. To define the localization of NHE9 at inhibitory synapses in response to neuronal activity. The hypothesis is that NHE9 is at the sub-surface of inhibitory synapses, where it is involved in trafficking receptors via the endocytic pathway. Genetic analyses show relationships between NHE9 and GABAergic receptor subunits. Immunocytochemistry of primary neurons will confirm these relationships and define the synaptic location of NHE9. Aim 3. To determine the role of NHE9 in developmental control of GABAergic synapse number and circuit properties in hippocampus. The hypothesis is that loss of NHE9 will over-acidify endosomes and impair proper trafficking of receptors, resulting in perturbation of E:I balance. Genetic studies reveal relationships between NHE9 and genes involved in inhibitory synapse development. Analysis of postsynaptic protein surface expression and physiological studies assessing overall E:I ratio should demonstrate alterations as a result of manipulation of NHE9 levels. NHE9 represents a new cellular mechanism in autism. The overall goal of the study is to provide treatment-facilitating insight into the pathology of a genetic form of autism.