The Role of Cation/Proton Exchanger NHE9 in Synaptic Transmission & Autism

Autism spectrum disorders (ASD) are neurodevelopmental syndromes that affect an estimated 1.5 million people in the United States alone. While ASDs arising from single gene mutations are rare, the study of these conditions provides a powerful and effective approach toward understanding the molecular basis for ASD as well as normal social behaviors. Recently, the cation/hydrogen exchanger Nhe9 has surfaced as a locus for inherited autism, with mutations confirmed to abolish expression. In addition, gene expression analysis from thousands of idiopathic ASD patients has shown altered Nhe9 gene expression as a more general feature of ASD. Interestingly, Nhe9 has also been identified as the single most significant locus for attention deficit hyperactivity disorder. Multiple lines of evidence thus implicate Nhe9 in the development of normal social behavior. In non-neural cells, NHE9 localizes to late endosomes where it mediates a H+ leak from these organelles, thus increasing their pH, and loss of NHE9 from these cells has been shown to hyper-acidify the vesicles. However, to date nothing is known about the function of NHE9 in neurons. This work seeks to understand the neurobiology of ASDs by investigating NHE9 knockout mice at behavioral, synaptic and cellular levels./nTo understand the role of NHE9 in autism, we created a conditional KO mouse lacking Nhe9 only in the central nervous system. The first goal of the study was to determine whether these animals exhibit behaviors analogous to human autism. Using a wide range of behavioral assays we determined that NHE9 cKO mice have altered sociability, repetitive behaviors, and impaired olfactory communication making them a suitable model for autism in mice. Immunostaining of cultured neurons localized NHE9 to both dendritic and axonal endosomes. As a protein of known biochemical function, we hypothesized that loss of NHE9 from these endosomal vesicles would result in over-acidification of those compartments, and indeed NHE9 KO neuron endosomes are more acidic than their WT counterparts. The physiological consequence of this hyper-acidification was investigated through electrophysiological recordings in acute hippocampal slices. These measurements revealed that loss of NHE9 impairs transmitter release by a reduction in release probability and decreased quantal size. Further investigation into the nature of this impairment, using VGLUT1-pHluorin imaging, discovered that hyper-acidic synaptic vesicles have a reduced rate of exocytosis and that acute neutralization of the pH gradient rescues this impairment. While it has yet to be determined how vesicular pH directly influences vesicle exocytosis, these findings reveal a new role for ΔpH in synaptic vesicle biology. Vesicles that are too acidic result in reduced excitatory transmission and yet dissipation of the proton gradient will collapse the driving forces required for retention of glutamate in the vesicles and abolish synaptic transmission. Thus, this study highlights the importance of the finely tuned balance in the proton-electrochemical gradient inside synaptic vesicle and the potential challenge of targeting this system to treat ASDs.

Advisor: Edwards, Robert H
Isacoff, Ehud Y